ElevateBio Partners with the California Institute for Regenerative Medicine to Accelerate the Development of Regenerative Medicines – Yahoo Finance

- ElevateBio to enable access to multiple induced pluripotent stem cell (iPSC) lines suitable for research through clinical development and commercialization

- ElevateBio to offer end-to-end development and GMP manufacturing capabilities to bring concepts to commercialization for regenerative medicines

WALTHAM, Mass., August 23, 2022--(BUSINESS WIRE)--ElevateBio, LLC (ElevateBio), a technology-driven company focused on powering transformative cell and gene therapies, today announced that it has partnered with the California Institute for Regenerative Medicine (CIRM) to advance the discovery and development of regenerative medicine as part of CIRMs Industry Alliance Program. Through the partnership, ElevateBio will provide access to high quality, well-characterized iPSC lines to academic institutions and biopharmaceutical companies that are awarded CIRM Discovery and Translational Grants. ElevateBio will also offer access to its viral vector technology, process development, analytical development, and Good Manufacturing Practice (GMP) manufacturing capabilities that are part of its integrated ecosystem built to power the cell and gene therapy industry.

"This exciting partnership with CIRM reflects the novelty of our iPSC platform and recognition of our next-generation cell lines that address industry challenges and could potentially save time and costs for partners developing iPSC-derived therapeutics," said David Hallal, Chairman and Chief Executive Officer of ElevateBio. "We are setting a new standard with iPSCs that can streamline the transition from research to clinical development and commercialization and leveraging our unique ecosystem of enabling technologies and expertise to help strategic partners harness the power of regenerative medicines."

With $5.5 billion in funding from the state of California, CIRM has funded 81 clinical trials and currently supports over 161 active regenerative medicine research projects spanning candidate discovery through phase III clinical trials. As part of CIRMs expansion of its Industry Alliance Program to incorporate Industry Resource Partners, this partnership will provide CIRM Awardees the option to license ElevateBios iPSC lines produced in xeno-free, feeder-free conditions using non-integrating technologies and have the ability to gain access to other enabling technologies, including gene editing, cell and vector engineering, and end-to-end services within ElevateBios integrated ecosystem, which are essential for driving the development of regenerative medicines.

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About ElevateBio:

ElevateBio is a technology-driven company built to power the development of transformative cell and gene therapies today and for many decades to come. The company has assembled industry-leading talent, built state-of-the-art facilities, and integrated diverse technology platforms, including gene editing, induced pluripotent stem cells (iPSCs), and protein, vector, and cellular engineering, necessary to drive innovation and commercialization of cellular and genetic medicines. In addition, BaseCamp is a purpose-built facility offering process innovation, process sciences, and current Good Manufacturing Practice (cGMP) manufacturing capabilities. Through BaseCamp and its enabling technologies, ElevateBio is focused on growing its collaborations with industry partners while also developing its own portfolio of cellular and genetic medicines. ElevateBio's team of scientists, drug developers, and company builders are redefining what it means to be a technology company in the world of drug development, blurring the line between technology and healthcare.

ElevateBio is located in Waltham, Mass. For more information, visit us at http://www.elevate.bio, or follow Elevate on LinkedIn, Twitter, or Instagram.

View source version on businesswire.com: https://www.businesswire.com/news/home/20220823005087/en/

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Media contact: Courtney Heath ScientPR Courtney@scientpr.com

Excerpt from:
ElevateBio Partners with the California Institute for Regenerative Medicine to Accelerate the Development of Regenerative Medicines - Yahoo Finance

Repeated intravenous administration of hiPSC-MSCs enhance the efficacy of cell-based therapy in tissue regeneration | Communications Biology -…

The therapeutic efficacy of intravenous hiPSC-MSCs infusion without intramuscular cellular transplantation

First, we determined whether hiPSC-MSCs could migrate into the ischemic limb after a single intravenous cellular infusion. Our results showed that most of the hiPSC-MSCs engrafted into the liver 12h after infusion (Supplementary Fig.1). The engrafted hiPSC-MSCs gradually migrated into the ischemic limb at day 3 and disappeared at day 14 (Supplementary Fig.1). A few cells engrafted in the ischemic limb, the engraftment rate was extremely low, evidenced by the DiR signal that was 9.8106 at day 7 after a single intravenous administration of 5105 hiPSC-MSCs versus 1.4109 7 days after a single intramuscular injection.

To compare intravenous cellular administration and intramuscular cellular delivery, three groups of mice that received intravenous hiPSC-MSC infusion once, every week or every 3 days without intramuscular administration of hiPSC-MSCs respectively and one group that received intramuscular hiPSC-MSC delivery only were employed (Fig.1a). Intravenous administration of hiPSC-MSCs once, every week or every 3 days without intramuscular administration of hiPSC-MSCs in the Saline-MSC/once, Saline-MSC/week and Saline-MSC/3 days groups significantly improved blood perfusion from day 7 onwards compared with the ischemia group (Fig.1b, all p<0.05). Repeated intravenous administration of hiPSC-MSCs in the Saline-MSC/week and Saline-MSC/3 days groups further increased blood perfusion at day 35 compared with the Saline-MSC/once group (Fig.1b, all p<0.05), although there was no difference between the first two groups (Fig.1b, p>0.05). Nevertheless intramuscular administration of hiPSC-MSCs in the MSC-Saline group achieved a better beneficial effect than intravenous administration of hiPSC-MSCs in the Saline-MSC/once, Saline-MSC/week and Saline-MSC/3 days groups from day 21 onwards (Fig.1b, all p<0.05).

To evaluate blood perfusion in the groups that received intravenous hiPSC-MSCs infusion without intramuscular hiPSC-MSCs transplantation, Laser Doppler imaging analysis was performed immediately and every week following femoral artery ligation (a). A single or repeated intravenous administration of hiPSC-MSCs in the Saline-MSC/once, Saline-MSC/week or Saline-MSC/3 days groups significantly increased blood perfusion from day 7 onwards compared with the ischemia group. Moreover, repeated intravenous hiPSC-MSCs infusion further improved blood perfusion at day 35. Nonetheless intramuscular hiPSC-MSC transplantation in the MSC-Saline group showed a superior beneficial effect over repeated intravenous hiPSC-MSC infusion in the Saline-MSC/week and Saline-MSC/3 days groups (b).

Taken together, our results demonstrated that systemic intravenous administration of hiPSC-MSCs without intramuscular administration of hiPSC-MSCs improved blood perfusion. Repeated intravenous administration of hiPSC-MSCs every week or every 3 days without intramuscular administration of hiPSC-MSCs further increased blood perfusion compared with a single intravenous injection, although there was no significant difference between intravenous administration repeated every week versus every 3 days. Nonetheless intramuscular administration of hiPSC-MSCs achieved a better beneficial effect than intravenous administration of hiPSC-MSCs once, every week or every 3 days.

Five groups of ICR mice were employed in the main experiment (Fig.2): (1) ischemia group receiving intravenous administration of saline immediately after induction of ischemia and intramuscular administration of culture medium at day 7; (2) MSC-Saline group receiving intravenous administration of saline immediately after induction of ischemia and intramuscular administration of 3106 hiPSC-MSCs at day 7; (3) MSC-MSC/once group receiving intravenous administration of 5105 hiPSC-MSCs immediately after induction of ischemia and intramuscular administration of 3106 hiPSC-MSCs at day 7; (4) MSC-MSC/week group receiving repeated intravenous administration of 5105 hiPSC-MSCs immediately and every week following induction of ischemia for 4 weeks and intramuscular administration of 3106 hiPSC-MSCs at day 7; (5) MSC-MSC/3 days group receiving repeated intravenous administration of 5105 hiPSC-MSCs immediately and every 3 days following induction of ischemia for 4 weeks and intramuscular administration of 3106 hiPSC-MSCs at day 7.

There are five groups of ICR mice in main experiment: ischemia group, MSC-Saline group, MSC-MSC/once group, MSC-MSC/week group, MSC-MSC/3 days group.

Serial laser doppler imaging and analysis was performed to evaluate the blood perfusion and monitor the blood flow recovery in the ischemic hind limb (Fig.3a). After induction of ischemia, blood perfusion of the ligated limb significantly decreased to an extremely low level relative to the non-ligated limb in the ischemia group (2.980.56), MSC-Saline group (2.960.30), MSC-MSC/once group (2.950.48), MSC-MSC/week group (3.010.29) and MSC-MSC/3 days group (2.970.30). There was no significant difference between the five groups (Fig.3b, all p>0.05). These results confirmed that acute hind-limb ischemia was induced in all groups. Intramuscular administration of hiPSC-MSCs with intravenous administration of saline or with intravenous administration of hiPSC-MSCs once or every week or every 3 days in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups resulted in a significant and progressive improvement in the blood perfusion of the ligated limb from day 14 onwards compared with the ischemia group (Fig.3b, all p<0.05). Intravenous administration of hiPSC-MSCs significantly increased blood perfusion in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups from day 7 onwards compared with the ischemia and MSC-Saline groups (Fig.3b, all p<0.05). Repeated intravenous administration of hiPSC-MSCs in the MSC-MSC/week and MSC-MSC/3 days groups further increased blood perfusion from day 28 onwards compared with the MSC-MSC/once group (Fig.3b, all p<0.05). Nevertheless there was no significant difference between mice that received repeated intravenous administration of hiPSC-MSCs in the MSC-MSC/week versus MSC-MSC/3 days groups throughout the study period. On day 35, blood perfusion of the ligated hind limb in the ischemia, MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups were 30.570.81, 40.560.84, 44.990.75, 50.410.68 and 51.120.86 respectively.

Laser Doppler imaging analysis was performed immediately and every week following femoral artery ligation to evaluate blood perfusion in the ischemic hind limbs (a). After intramuscular transplantation of hiPSC-MSCs, blood perfusion was significantly improved in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups compared with the ischemia group from day 14 onwards (all p<0.05). A single and repeated intravenous hiPSC-MSC infusion further improved blood perfusion in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups compared with MSC-Saline group (all p<0.05). Moreover, the blood perfusion was significantly higher in the MSC-MSC/week and MSC-MSC/3 days groups compared with the MSC-MSC/once group (all p<0.05). There was no significant difference between the MSC-MSC/week and MSC-MSC/3 days groups (p>0.05) (b).

Taken together, our results showed that systemic intravenous administration of hiPSC-MSCs combined with intramuscular transplantation of hiPSC-MSCs improved blood perfusion in a mouse model of hind-limb ischemia relative to intramuscular hiPSC-MSC transplantation without systemic hiPSC-MSC delivery. In addition, repeated intravenous administration of hiPSC-MSCs every week or every 3 days further improved the therapeutic effects of hiPSC-MSC-based therapy compared with a single intravenous injection. No significant difference was observed between repeated intravenous administration of hiPSC-MSCs every week and every 3 days.

To evaluate neovascularization in the ischemic limb, immunohistochemical staining with anti-mouse alpha-smooth muscle antigen (-SMA) and anti-mouse von Willebrand factor (vWF) antibodies were performed to assess arteriogenesis and angiogenesis following cellular transplantation respectively (Fig.4a). On day 14, intramuscular transplantation of hiPSC-MSCs in the MSC-Saline group did not increase arteriogenesis and capillary formation (Fig.4b,c, p>0.05). Nevertheless, systemic intravenous administration of hiPSC-MSCs in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly improved arteriogenesis and capillary formation compared with the ischemia group (Fig.4b,c, all p<0.05). On day 35, compared with the ischemia group, intramuscular transplantation of hiPSC-MSCs in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly increased neovascularization (Fig.4b,c, all p<0.05). Moreover, systemic intravenous administration of hiPSC-MSCs in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups further improved neovascularization compared with the MSC-Saline group on day 35 (Fig.4b,c, p<0.05). In addition, repeated intravenous administration of hiPSC-MSCs in the MSC-MSC/week and MSC-MSC/3 days groups further promoted neovascularization compared with the MSC-MSC/once group (Fig.4b,c, all p<0.05). There was no difference in neovascularization between the MSC-MSC/week and MSC-MSC/3 days groups (Fig.4b,c, all p>0.05).

Immunohistochemical staining with anti-mouse vWF (green) and anti-mouse -SMA (red) antibodies was performed to assess angiogenesis and arteriogenesis in ischemic tissues. Massons trichrome staining was performed to evaluate the degree of fibrosis (a). On day 14, neovascularization was markedly increased in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups, not in the MSC-Saline group, relative to the ischemia group. On day 35, after intramuscular transplantation of hiPSC-MSCs, neovascularization was significantly improved in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups compared with the ischemia group (all p<0.05). Intravenous administration of hiPSC-MSCs in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups enhanced the therapeutic effects of intramuscularly transplanted hiPSC-MSCs on neovascularization compared with the MSC-Saline group (all p<0.05). Moreover, neovascularization was further enhanced by repeated intravenous hiPSC-MSC infusion in the MSC-MSC/week and MSC-MSC/3 days groups compared with the MSC-MSC/once group (b, c). On day 14, fibrosis was remarkably decreased in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups, not in the MSC-Saline group, relative to the ischemia group. On day 35, after intramuscular transplantation of hiPSC-MSCs, fibrosis was significantly reduced in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups compared with the ischemia group (all p<0.05). Intravenous administration of hiPSC-MSCs in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups enhanced the therapeutic effects of intramuscularly transplanted hiPSC-MSCs on reduction of fibrosis compared with the MSC-Saline group (all p<0.05). Moreover, the anti-fibrotic effect was further enhanced by repeated intravenous hiPSC-MSC infusion in the MSC-MSC/week and MSC-MSC/3 days groups compared with the MSC-MSC/once group (d).

To assess the degree of fibrosis in the ischemic limb, Massons Trichrome staining were performed to determine the percentage of fibrotic tissue in the ischemic limb (Fig.4a). On day 14, intramuscular transplantation of hiPSC-MSCs in the MSC-Saline group did not decrease fibrosis (Fig.4d, p>0.05). Nevertheless, systemic intravenous administration of hiPSC-MSCs in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly reduced fibrosis compared with the ischemia group (Fig.4d, all p<0.05). Compared with the ischemia group, intramuscular transplantation of hiPSC-MSCs in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly ameliorated fibrosis on day 35 (Fig.4d, all p<0.05). Moreover, systemic intravenous administration of hiPSC-MSCs in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly reduced fibrosis compared with the MSC-Saline group (Fig.4d, all p<0.05). In addition, repeated intravenous administration of hiPSC-MSCs in the MSC-MSC/week and MSC-MSC/3 days groups further decreased fibrosis compared with the MSC-MSC/once group (Fig.4d, all p<0.05). There were no differences in fibrosis between the MSC-MSC/week and MSC-MSC/3 days groups (Fig.4d, all p>0.05).

Taken together, our results showed that systemic intravenous administration of hiPSC-MSCs combined with intramuscular transplantation of hiPSC-MSCs promoted neovascularization and reduced fibrosis in a mouse model of hind-limb ischemia. Repeated intravenous administration of hiPSC-MSCs every week or every 3 days further increased the neovascularization and decreased the fibrosis following cellular transplantation compared with a single intravenous injection. No significant difference was observed between repeated intravenous administration of hiPSC-MSCs every week and every 3 days.

Fluorescent imaging of ischemic hind limbs was performed immediately and every week after induction of ischemia to access the cellular engraftment and survival of intramuscularly transplanted hiPSC-MSCs (Fig.5a). To avoid any confusion on the fluorescent signal, intravenous administered hiPSC-MSCs were not labeled with DiR. There was no significant difference in fluorescent signal intensity over the ischemic hind limb after intramuscular cellular transplantation (Fig.5b, all p>0.05). Systemic intravenous administration of hiPSC-MSCs significantly increased cellular engraftment and survival in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups from day 14 onwards relative to the MSC-Saline group (Fig.5b, all p<0.05). Moreover, repeated intravenous administration of hiPSC-MSCs in the MSC-MSC/week and MSC-MSC/3 days groups further improved cellular engraftment and survival from day 21 onwards compared with the MSC-MSC/once group (Fig.5b, all p<0.05). There was no significant difference between mice that received repeated intravenous administration of hiPSC-MSCs in the MSC/week and MSC-MSC/3 days groups throughout the study period. On day 35, the estimated survival rates in MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups decreased to 2.590.31%, 8.330.54%, 13.560.49% and 14.230.42%, respectively (Supplementary Fig.2 and Supplementary Data1).

A series of fluorescent images of ischemic hind limbs was performed immediately and every week following intramuscular transplantation of hiPSC-MSCs to detect the fate of intramuscularly transplanted hiPSC-MSCs (a). A single or repeated intravenous hiPSC-MSCs infusion in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly prolonged the survival of intramuscular transplanted hiPSC-MSCs from day 14 onwards compared with the MSC-Saline group (all p<0.05). Moreover, repeated intravenous hiPSC-MSCs infusion in the MSC-MSC/week and MSC-MSC/3 days groups further improved the survival of intramuscularly transplanted hiPSC-MSCs from day 21 onwards compared with the MSC-MSC/once group (all p<0.05), whereas no significant difference was observed between MSC-MSC/week and MSC-MSC/3 days groups (p>0.05) (b).

Cellular engraftment and survival of intramuscularly transplanted hiPSC-MSCs were further confirmed by immunohistochemical double staining with anti-human GAPDH and anti-human mitochondria antibodies (Fig.6a). Systemic intravenous administration of hiPSC-MSCs significantly increased human GAPDH and human mitochondria positive cells over the ischemic hind limb in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups from day 14 onwards relative to the MSC-Saline group (Fig.6b, all p<0.05). Moreover, on day 35, repeated intravenous administration of hiPSC-MSCs in the MSC-MSC/week and MSC-MSC/3 days groups further increased the human GAPDH and human mitochondria positive cells compared with the MSC-MSC/once group (Fig.6b, all p<0.05). No difference between the MSC-MSC/week and MSC-MSC/3 days groups was noted (Fig.6b, all p>0.05).

The engraftment of intramuscularly transplanted hiPSC-MSCs was further confirmed by double immunohistochemical staining with anti-human GAPDH (green) and anti-human mitochondria antibodies (red) at day 14 and 35 (a). A single or repeated intravenous hiPSC-MSC infusion in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly improved the engraftment of intramuscularly transplanted hiPSC-MSCs from day 14 onwards (all p<0.05). Repeated intravenous hiPSC-MSC infusion in the MSC-MSC/week and MSC-MSC/3 days groups further improved the engraftment of intramuscular transplanted hiPSC-MSCs at day 35 compared with the MSC-MSC/once group (all p<0.05), whereas no significant difference was observed between the MSC-MSC/week and MSC-MSC/3 days groups (p>0.05) (b).

Taken together, our results demonstrated that systemic intravenous administration of hiPSC-MSCs enhanced engraftment and survival of intramuscularly transplanted hiPSC-MSCs. In addition, repeated intravenous administration every week or every 3 days further increased the cellular engraftment and survival compared with a single intravenous injection. No significant difference was observed between repeated intravenous administration of hiPSC-MSCs every week versus every 3 days.

Immunohistochemical staining with anti-mouse CD68 antibody was performed to calculate the number of macrophages after cellular transplantation and evaluate the infiltration of macrophages (Fig.7a). M2 macrophages were further characterized by immunohistochemical staining with anti-mouse Arginase-1 antibody (Fig.7a). Although there was no significant difference between any of the five groups at day 7 and 14 after induction of ischemia (Fig.7b, all p>0.05), intramuscular administration of hiPSC-MSCs in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly increased M2 macrophage polarization in the ligated limb from day 14 onwards relative to the ischemia group (Fig.7c, all p<0.05). Moreover, intravenous administration of hiPSC-MSCs remarkedly promoted M2 macrophage polarization in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups from day 7 onwards compared with the ischemia and MSC-Saline groups (Fig.7c, all p<0.05). On day 35, intramuscular administration of hiPSC-MSCs in MSC-Saline group had significantly decreased the infiltration of macrophages although the M2 macrophage percentage was similar to that in the ischemia group (Fig.7b,c, all p<0.05). Systemic intravenous administration of hiPSC-MSCs in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly decreased macrophage infiltration and increased M2 macrophage polarization relative to the MSC-Saline group (Fig.7b,c, all p<0.05). Repeated intravenous administration of hiPSC-MSCs in the MSC-MSC/week and MSC-MSC/3 days groups further reduced the infiltration of macrophages and increased the polarization of M2 macrophages compared with the MSC-MSC/once group (Fig.7b,c, all p<0.05). There was no noticeable difference in either the infiltration of macrophages or polarization of M2 macrophages between the MSC-MSC/week and MSC-MSC/3 days groups (Fig.7b,c, all p>0.05).

Muscular infiltration of macrophages was determined by immunohistochemical staining with anti-mouse CD68 antibody (green) at day 7, 14, and 35. Number of M2 macrophages was detected by immunohistochemical staining with anti-mouse Arginase-1 antibodies (red) (a). At day 35, after intramuscular transplantation of hiPSC-MSCs, total macrophages were significantly decreased in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups compared with the ischemia group (all p<0.05). A single or repeated intravenous hiPSC-MSCs infusion in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly decreased the muscular infiltration of macrophages compared with the MSC-Saline group (all p<0.05). In addition, repeated intravenous hiPSC-MSCs infusion in the MSC-MSC/week and MSC-MSC/3 days groups further decreased the muscular infiltration of macrophages compared with the MSC-MSC/once group (all p<0.05). Nevertheless no significant difference was observed between groups at day 7 and 14 (all p>0.05) (b). Intramuscular transplantation of hiPSC-MSCs without intravenous hiPSC-MSC infusion significantly increased the polarization of M2 macrophages at day 14 compared with the ischemia group (p<0.05). A single or repeated intravenous hiPSC-MSC infusion in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly improved the polarization of M2 macrophages from day 7 onwards (all p<0.05). Repeated hiPSC-MSCs infusion further promoted the polarization of M2 macrophages compared with a single intravenous hiPSC-MSCs infusion in the MSC-MSC/once group at day 35 (all p<0.05) (c).

Taken together, our results demonstrated that systemic intravenous administration of hiPSC-MSCs decreased the infiltration of macrophages and increased the polarization of M2 macrophages. Repeated intravenous administration of hiPSC-MSCs every week or every 3 days further decreased the infiltration of macrophages and increased the polarization of M2 macrophages compared with a single intravenous injection, whereas no significant difference was observed between repeated intravenous administration of hiPSC-MSCs every week and every 3 days.

The limb tissue level of a specific subset-related cytokines was measured using a commercial mouse inflammatory factor array. For anti-inflammatory cytokines, on day 14, there was no significant difference on interleukin (IL)10 and vascular endothelial growth factor (VEGF) among the ischemia, MSC-Saline and MSC-MSC/once groups (Supplementary Fig.3a,b, all p>0.05). Nonetheless, repeated systemic intravenous hiPSC-MSC infusion in the MSC-MSC/week and MSC-MSC/3 days groups significantly increased IL-10 and VEGF compared with the ischemia group (Supplementary Fig.3a,b, all p<0.05). Moreover, an increase of IL-10 was observed in the MSC-MSC/week and MSC-MSC/3 days groups relative to the MSC-Saline group (Supplementary Fig.3a,b, all p<0.05). On day 35, intramuscular transplantation of hiPSC-MSCs in the MSC-Saline group did not significantly improved IL-10 relative to ischemia group. Nevertheless, systemic intravenous hiPSC-MSC infusion in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly improved IL-10 compared with the ischemia group (Supplementary Fig.3a, all p<0.05). Moreover, repeated systemic intravenous hiPSC-MSC infusion in the MSC-MSC/week and MSC-MSC/3 days groups further increased IL-10 compared with the MSC-MSC/once group (Supplementary Fig.3a, all p<0.05). No significant difference on VEGF was observed among all five groups on day 35 (Supplementary Fig.3b, all p<0.05).

For inflammatory cytokines, on day 14, intramuscular transplantation of hiPSC-MSCs in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly decreased IL-1A and IL-17A compared with the ischemia group (Supplementary Fig.3c,d, all p<0.05). Nonetheless, there was no significant difference among the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups (Supplementary Fig.3c,d, all p>0.05). There was no significant difference on IL-2 and macrophage colony-stimulating factor (MCSF) among the ischemia, MSC-Saline and MSC-MSC/once groups (Supplementary Fig.3e,f, all p>0.05). Nonetheless, repeated systemic intravenous hiPSC-MSC infusion in the MSC-MSC/week and MSC-MSC/3 days groups significantly decreased IL-2 and MCSF compared with the ischemia group (Supplementary Fig.3e,f, all p<0.05). On day 35, intramuscular transplantation of hiPSC-MSCs in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly reduced IL-17A relative to ischemia group (Supplementary Fig.3d, all p<0.05). Moreover, repeated systemic intravenous hiPSC-MSC infusion in the MSC-MSC/week and MSC-MSC/3 days groups further decreased IL-17A compared with the MSC-Saline and MSC-MSC/once groups respectively (Supplementary Fig.3d, all p<0.05). No significant difference on IL-1A, IL-2 and MCSF was observed among all five groups on day 35 (Supplementary Fig.3c,e,f, all p>0.05).

Taken together, our results demonstrated that systemic intravenous administration of hiPSC-MSCs could improve anti-inflammatory cytokines and decreased inflammatory cytokines. Repeated intravenous administration of hiPSC-MSCs every week or every 3 days further improved anti-inflammatory cytokines and decreased inflammatory cytokines compared with a single intravenous injection. No significant difference was observed between repeated intravenous administration of hiPSC-MSCs every week and every 3 days.

Flow cytometry analysis of fresh splenocytes was performed to assess splenic Tregs and natural killer (NK) cells populations and so determine the in vivo immunomodulatory effect of systemic administration of hiPSC-MSCs (Fig.8a). Splenic NK cells were defined as both a CD49b-FITC and NK1.1-APC positive cell population. Our result showed that splenic NK cells progressively decreased following intramuscular hiPSC-MSC transplantation or intravenous hiPSC-MSC infusion in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups, whereas no significant difference was noted between different time points in the ischemia group (Supplementary Fig.4a). Compared with the ischemia group, intramuscular administration of hiPSC-MSCs in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly decreased splenic NK cells from day 14 onwards (Fig.8b, all p<0.05). Systemic intravenous hiPSC-MSC infusion in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly reduced splenic NK cells from day 7 onwards relative to the ischemia and MSC-Saline groups (Fig.8b, all p<0.05). Repeated systemic intravenous hiPSC-MSC infusion in the MSC-MSC/week and MSC-MSC/3 days groups further reduced splenic NK cells from day 14 onwards compared with the MSC-MSC/once group (Fig.8b, all p<0.05). Nonetheless no significant difference was observed between the MSC-MSC/week and MSC-MSC/3 days groups (Fig.8b, all p>0.05).

Splenic Tregs and NK cells were determined by flow cytometry analysis at day 7, 14 and 35 (a). After intramuscular transplantation of hiPSC-MSCs, splenic NK cells were significantly decreased in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups from day 14 onwards compared with the ischemia group (all p<0.05). A single or repeated intravenous hiPSC-MSC infusion in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly decreased splenic NK cells from day 7 onwards compared with the ischemia and MSC-Saline groups (all p<0.05). Repeated intravenous hiPSC-MSC infusion in the MSC-MSC/week and MSC-MSC/3 days groups further decreased splenic NK cells from day 14 onwards compared with the MSC-MSC/once group (all p<0.05) (b). After intramuscular transplantation of hiPSC-MSCs, splenic Tregs were significantly increased in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups at day 35 compared with the ischemia group (all p<0.05). A single or repeated intravenous hiPSC-MSC infusion in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly increased splenic Tregs compared with the ischemia and MSC-Saline groups (all p<0.05). Moreover, repeated intravenous hiPSC-MSC infusion in the MSC-MSC/week and MSC-MSC/3 days groups further increased splenic Tregs from day 14 onwards compared with the MSC-MSC/once group (all p<0.05) (c).

Splenic Tregs were determined as Foxp3 positive cells in a proportion of pre-gated CD4 positive cells. Our result showed that splenic Tregs reached a peak on day 7 in the MSC-MSC/once group, whereas these immunomodulatory cells continued to increase in the MSC-MSC/week and MSC-MSC/3 days groups. No significant difference was observed between different time points in the ischemia and MSC-Saline groups (Supplementary Fig.4b). Compared with the ischemia group, intramuscular administration of hiPSC-MSCs in the MSC-Saline, MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly increased splenic Tregs on day 35 (Fig.8c, all p<0.05). Intravenous hiPSC-MSC infusion in the MSC-MSC/once, MSC-MSC/week and MSC-MSC/3 days groups significantly improved splenic Tregs from day 7 onwards compared with the ischemia and MSC-Saline groups (Fig.8c, all p<0.05). Repeated systemic intravenous hiPSC-MSCs infusion in the MSC-MSC/week and MSC-MSC/3 days groups further increased splenic Tregs from day 14 onwards compared with the MSC-MSC/once group (Fig.8c, all p<0.05), but there was no significant difference between the MSC-MSC/week and MSC-MSC/3 days groups (Fig.8c, all p>0.05).

Taken together, our results demonstrated that systemic intravenous administration of hiPSC-MSCs could modulate systemic immune cell activation by decreasing splenic NK cells as well as increasing splenic Tregs. Repeated intravenous administration of hiPSC-MSCs every week or every 3 days further decreased splenic NKs and increased splenic Tregs compared with a single intravenous injection. No significant difference was observed between repeated intravenous administration of hiPSC-MSCs every week and every 3 days.

To compare the survival and engraftment of intramuscularly transplanted hiPSC-MSCs with intervenous infusion of hiPSC-MSCs and subcutaneous administration of cyclosporine A, fluorescent imaging of ischemic hind limb was performed immediately and every week in the MSC-Saline-Cyc, MSC-MSC/once-Cyc and MSC-MSC/week-Cyc groups (Supplementary Fig.5a). There was no significant difference in cellular engraftment between the MSC-MSC/once and MSC-Saline-Cyc groups through this study (Supplementary Fig.5b, p>0.05). Although repeated intravenous infusion of hiPSC-MSCs without subcutaneous administration of cyclosporine A remarkedly increased cell engraftment in the MSC-MSC/week group relative to the MSC-MSC/once group (Supplementary Fig.5b, p<0.05), no significant difference was observed after subcutaneous administration of cyclosporine A between the MSC-MSC/week-Cyc and MSC-MSC/once-Cyc groups (Supplementary Fig.5b, p>0.05). Nonetheless, subcutaneous administration of cyclosporine A did not improve the cell engraftment in the MSC-MSC/once-Cyc and MSC-MSC/week-Cyc groups relative to the MSC-MSC/once and MSC-MSC/week groups respectively (Supplementary Fig.5b, p>0.05).

To compare the therapeutic efficacy of intramuscularly transplanted hiPSC-MSCs with intervenous infusion of hiPSC-MSCs and subcutaneous administration of cyclosporine A, serial laser doppler imaging and analysis was performed to evaluate the blood perfusion and monitor the blood flow recovery in the ischemic hind limb (Supplementary Fig.6a). When comparison between the MSC-MSC/once and MSC-Saline-Cyc groups was performed, intravenous infusion of hiPSC-MSCs significantly improved blood perfusion in the MSC-MSC/once group relative to MSC-Saline-Cyc group during the first 2 weeks (Supplementary Fig.6b, p<0.05). Following intramuscular hiPSC-MSC transplantation at day 7, blood perfusion progressly increased in the MSC-MSC/once and MSC-Saline-Cyc groups. Nevertheless, no significant difference was observed between the MSC-MSC/once and MSC-Saline-Cyc groups from day 21 onwards (Supplementary Fig.6b, p>0.05). Repeated intravenous infusion of hiPSC-MSCs with or without subcutaneous administration of cyclosporine A significantly improved blood perfusion at day 35 in the MSC-MSC/week and MSC-MSC/week-Cyc groups compared with the MSC-MSC/once and MSC-MSC/once-Cyc groups respectively (Supplementary Fig.6b, p<0.05). Nonetheless, subcutaneous administration of cyclosporine A did not improve the blood perfusion in the MSC-MSC/once-Cyc and MSC-MSC/week-Cyc groups relative to the MSC-MSC/once and MSC-MSC/week groups respectively (Supplementary Fig.6b, p>0.05).

Cumulatively, our results demonstrated that no significant difference was observed in cell engraftment between a single or repeated intravenous hiPSC-MSC infusion and subcutaneous administration of cyclosporine A. Although there was no significant difference in blood perfusion between the cyclosporine A and single hiPSC-MSC infusion, a significantly improved blood perfusion was observed in the repeated hiPSC-MSC infusion groups relative to the cyclosporine A group. Furthermore, subcutaneous administration of cyclosporine A did not further increased cell engraftment or therapeutic efficacy in either single or repeated hiPSC-MSC infusion groups.

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Repeated intravenous administration of hiPSC-MSCs enhance the efficacy of cell-based therapy in tissue regeneration | Communications Biology -...

Century Therapeutics Receives Study May Proceed Notification from FDA for CNTY-101, the First … – The Bakersfield Californian

Investigational New Drug Application for CNTY-101, a CAR-iNK product candidate targeting CD19 for B-cell malignancies, cleared by FDA

First cell product candidate engineered with six precision gene edits including a CD19-CAR, Allo-Evasiontechnology, IL-15 cytokine support and a safety switch

Phase 1 ELiPSE-1 trial evaluating CNTY-101 in relapsed or refractory CD19 positive B-cell malignancies anticipated to begin in 2H22

PHILADELPHIA, Aug. 25, 2022 (GLOBE NEWSWIRE) -- Century Therapeutics, Inc., (NASDAQ: IPSC), an innovative biotechnology company developing induced pluripotent stem cell (iPSC)-derived cell therapies in immuno-oncology, announced today that the company has been notified by the U.S. Food and Drug Administration (FDA) that the Companys ELiPSE-1 clinical study may proceed to assess CNTY-101 in patients with relapsed or refractory CD19 positive B-cell malignancies. CNTY-101 is the first allogeneic cell therapy product candidate engineered with four powerful and complementary functionalities, including a CD19 CAR for tumor targeting, IL-15 support for enhanced persistence, Allo-Evasiontechnology to prevent host rejection and enhance persistence and a safety switch to provide the option to eliminate the drug product if ever necessary. CNTY-101 is manufactured from a clonal iPSC master cell bank that yields homogeneous product, in which all infused cells have the intended modifications.

This IND clearance is a significant milestone for Century as we execute on our vision to merge two disruptive platforms, precision gene editing and the powerful potential of iPSCs, to potentially move the allogeneic cell therapy field forward, and continue on our path to becoming a leader in the space, said Lalo Flores, Chief Executive Officer, Century Therapeutics. We believe that CNTY-101, our first and wholly owned product candidate, will be the most technically advanced and differentiated CD19-targeted cell product when it enters the clinic, which is anticipated to occur later this year. We look forward to assessing the potential of Allo-Evasionto prevent immunological rejection and enhance persistence of multiple dosing of CNTY-101 regimens with the aim to increase the proportion of patients that achieve durable responses.

CNTY-101 is the first allogeneic cell product candidate with six genetic modifications incorporated using sequential rounds of CRISPR-mediated homologous recombination and repair that has received IND clearance by the FDA, said Luis Borges, Chief Scientific Officer, Century Therapeutics. We believe CNTY-101 will demonstrate the power of Centurys iPSC technology and cell engineering technology platforms. This accomplishment is a testament to the expertise and dedication of our team as we continue to make progress developing our pipeline of iPSC-derived NK and T cell product candidates.

The Phase 1 trial, ELiPSE-1 ( NCT05336409 ), is intended to assess the safety, tolerability, pharmacokinetics and preliminary efficacy of CNTY-101 in patients with relapsed or refractory CD19-positive B-cell malignancies. All patients will receive an initial standard dose of conditioning chemotherapy consisting of cyclophosphamide (300 mg/m2) and fludarabine (30mg/m2) for 3 days. Schedule A of the trial includes a single-dose escalation of CNTY-101 and subcutaneous IL-2. Schedule B will evaluate a three-dose schedule per cycle of CNTY-101. Patients who demonstrate a clinical benefit are eligible for additional cycles of treatment with or without additional lymphodepletion pending FDA consent. We anticipate initiation of the Phase 1 trial later this year.

About Allo-Evasion

Centurys proprietary Allo-Evasiontechnology is used to engineer cell therapy product candidates with the potential to evade identification by the host immune system so they can be dosed multiple times without rejection, enabling increased persistence of the cells during the treatment period and potentially leading to deeper and more durable responses. More specifically, Allo-Evasion1.0 technology incorporates three gene edits designed to avoid recognition by patient/host CD8+ T cells, CD4+ T cells and NK cells. Knockout of beta-2-microglobulin or 2m, designed to prevent CD8+ T cell recognition, knock-out of the Class II Major Histocompatibility Complex Transactivator, or CIITA, designed to prevent CD4+ T cell recognition, and knock-in of the HLA-E gene, designed to enable higher expression of the HLA-E protein to prevent killing of CNTY-101 cells by host NK cells. Allo-Evasiontechnology may allow the implementation of more flexible and effective repeat dosing protocols for off-the-shelf product candidates.

About CNTY-101

CNTY-101 is an investigational off-the-shelf cancer immunotherapy product candidate that utilizes iPSC-derived natural killer (NK) cells with a CD19-directed chimeric antigen receptor (CAR) and includes Centurys core Allo-Evasionedits designed to overcome the three major pathways of host versus graft rejection - CD8+ T cells, CD4+ T cells and NK cells. In addition, the product candidate is engineered to express IL-15 to provide homeostatic cytokine support, which has been shown pre-clinically to improve functionality and persistence. Further, to potentially improve safety, the iNK cells were engineered with an EGFR safety switch, and proof-of-concept studies have demonstrated that the cells can be quickly eliminated by the administration of cetuximab, an antibody against EGFR approved by the U.S. Food and Drug Administration (FDA) for certain cancers. Initiation of the Phase 1, ELiPSE-1 trial in relapsed or refractory CD19-positive B-cell malignancies in multiple centers in the United States is anticipated to begin in the second half of 2022.

About Century Therapeutics

Century Therapeutics, Inc. (NASDAQ: IPSC) is harnessing the power of adult stem cells to develop curative cell therapy products for cancer that we believe will allow us to overcome the limitations of first-generation cell therapies. Our genetically engineered, iPSC-derived iNK and iT cell product candidates are designed to specifically target hematologic and solid tumor cancers. We are leveraging our expertise in cellular reprogramming, genetic engineering, and manufacturing to develop therapies with the potential to overcome many of the challenges inherent to cell therapy and provide a significant advantage over existing cell therapy technologies. We believe our commitment to developing off-the-shelf cell therapies will expand patient access and provide an unparalleled opportunity to advance the course of cancer care. For more information on Century Therapeutics please visit https://www.centurytx.com/.

Forward-Looking Statements

This press release contains forward-looking statements within the meaning of, and made pursuant to the safe harbor provisions of, The Private Securities Litigation Reform Act of 1995. All statements contained in this press release, other than statements of historical facts or statements that relate to present facts or current conditions, including but not limited to, statements regarding our clinical development plans and timelines are forward-looking statements. These statements involve known and unknown risks, uncertainties and other important factors that may cause our actual results, performance, or achievements to be materially different from any future results, performance or achievements expressed or implied by the forward-looking statements. In some cases, you can identify forward-looking statements by terms such as may, might, will, should, expect, plan, aim, seek, anticipate, could, intend, target, project, contemplate, believe, estimate, predict, forecast, potential or continue or the negative of these terms or other similar expressions. The forward-looking statements in this presentation are only predictions. We have based these forward-looking statements largely on our current expectations and projections about future events and financial trends that we believe may affect our business, financial condition, and results of operations. These forward-looking statements speak only as of the date of this press release and are subject to a number of risks, uncertainties and assumptions, some of which cannot be predicted or quantified and some of which are beyond our control, including, among others: our ability to successfully advance our current and future product candidates through development activities, preclinical studies, and clinical trials; our ability to obtain FDA acceptance for our future IND submissions and commence clinical trials on expected timelines, or at all; our reliance on the maintenance of certain key collaborative relationships for the manufacturing and development of our product candidates; the timing, scope and likelihood of regulatory filings and approvals, including final regulatory approval of our product candidates; the impact of the COVID-19 pandemic, geopolitical issues and inflation on our business and operations, supply chain and labor force; the performance of third parties in connection with the development of our product candidates, including third parties conducting our future clinical trials as well as third-party suppliers and manufacturers; our ability to successfully commercialize our product candidates and develop sales and marketing capabilities, if our product candidates are approved; and our ability to maintain and successfully enforce adequate intellectual property protection. These and other risks and uncertainties are described more fully in the Risk Factors section of our most recent filings with the Securities and Exchange Commission and available at http://www.sec.gov. You should not rely on these forward-looking statements as predictions of future events. The events and circumstances reflected in our forward-looking statements may not be achieved or occur, and actual results could differ materially from those projected in the forward-looking statements. Moreover, we operate in a dynamic industry and economy. New risk factors and uncertainties may emerge from time to time, and it is not possible for management to predict all risk factors and uncertainties that we may face. Except as required by applicable law, we do not plan to publicly update or revise any forward-looking statements contained herein, whether as a result of any new information, future events, changed circumstances or otherwise.

For More Information:

Company: Elizabeth Krutoholow investor.relations@centurytx.com

Investors: Melissa Forst/Maghan Meyers century@argotpartners.com

Media: Joshua R. Mansbach century@argotpartners.com

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Century Therapeutics Receives Study May Proceed Notification from FDA for CNTY-101, the First ... - The Bakersfield Californian

Alzheimer’s: Could controlling the brain’s own clean-up crew help? – Medical News Today

In a recent study published inNature Neuroscience, scientists revealed a novel screening platform for characterizing genes that regulate specific microglial functions which may contribute to Alzheimers disease (AD).

Characterizing regulatory genes that cause microglia to switch from a healthy state to a diseased state, such as in the brains of individuals with AD and other neurodegenerative conditions, could help develop therapeutics that target these genes or the proteins encoded by these genes.

Since microglia are guardians of the brains homeostasis, it is important to identify specific drivers that lead to neuronal toxicity for therapeutic intervention. Our new CRISPR screening platform [] enables us to identify these drivers in a rapid, scalable manner. We already uncovered druggable targets that control microglia states, and the next steps would be to test these in relevant preclinical models. Dr. Li Gan, study co-author and neuroscientist at the Weill Cornell Medical College, speaking to Medical News Today

AD is the most common form of dementia, accounting for 60-80% of all dementia cases. Despite the advances in the understanding of AD, there is a lack of effective treatments for this neurodegenerative disease.

The accumulation of the misfolded beta-amyloid protein into clumps or plaques is one of the hallmarks of AD. A considerable amount of research has focused on mutations that lead to the abnormal processing of the beta-amyloid protein and, subsequently, its accumulation.

However, treatments targeting the pathways involved in the processing of beta-amyloid have not been successful.

Moreover, researchers have found that individuals with AD often do not show mutations in genes associated with the accumulation of the amyloid protein. In contrast, recent evidence suggests that individuals with AD often show deficits in the clearance or removal of misfolded beta-amyloid.

This may be due to the dysfunction of microglia, which are the primary immune cells in the brain. One of the functions of microglia includes phagocytosisa process involving the ingestion of dead cells, pathogens, and misfolded proteins to facilitate their removal.

There is growing evidence that the ability of microglia to remove the beta-amyloid protein may be impaired in AD. Microglia may also contribute to the development of AD by secreting inflammatory proteins and causing excessive removal of neurons and synapses, the links between neurons that allow them to communicate.

In addition to AD, there is evidence suggesting that microglia may also contribute to the development of other neurodegenerative disorders.

However, the molecular mechanisms underlying the wide array of functions performed by microglia in normal conditions and diseases such as AD are not well understood.

Functional genetic screening is a tool used for identifying genes that are involved in a specific cellular function. Such screens involve the inhibition or activation of a specific gene in a cell to assess whether the change in expression levels of that gene impacts a certain function of interest, such as cell proliferation.

In recent years, researchers have adapted the gene-editing tool known as CRISPR-Cas9 to identify genes involved in various diseases, including cancer. The advantages of the CRISPR screening platform include its higher sensitivity and greater reproducibility than previously used screening methods.

CRISPR-Cas9 consists of a small piece of RNA called a guide sequence and the enzyme Cas9. The guide RNA binds to the DNA region of interest, allowing Cas9 to bind and cleave the DNA at the targeted site.

In the present study, the researchers used a modified CRISPR-Cas9 system involving a deactivated Cas9 (dCas9) enzyme that does not cleave the DNA. Besides the deactivated Cas9 enzyme, the modified CRISPR-dCas9 platform also consists of proteins that can either upregulate or downregulate the gene of interestor in other words, turn them on and off.

Such CRISPR screens involve the delivery of the guide RNA to the cell with the help of a genetically engineered virus a viral vector. However, using viruses to deliver the guide RNA to mature microglia has been challenging.

To circumvent these difficulties, the researchers used induced pluripotent stem cells (iPSCs). IPSCs are derived by reprogramming adult cells from tissue such as skin, hair, or blood, into an embryonic state.

Similar to stem cells from the embryo, these iPSCs can mature to form any desired cell type, including neurons or microglia. The benefit of using cells derived from iPSCs is that they more closely resemble human cells than conventional cell lines.

Moreover, microglia from mice and humans differ in the molecules released during an immune response. Thus, microglia derived from human iPSCs represent a better model for understanding how genes regulate microglial functions.

In the present study, the researchers used induced pluripotent stem cell lines, which were modified to express genes encoding the CRISPR-dCas9 machinery. The CRISPR machinery in the iPSCs was, however, inactive and could be activated only in the presence of the antibiotic trimethoprim.

The researchers then used viral vectors to deliver guide RNAs to the iPSCs. The iPSCs used by the researchers were genetically engineered to rapidly differentiate or mature into microglia-like cells upon exposure to a specialized culture medium.

Upon differentiating the iPSCs into microglial cells, the researchers activated CRISPR machinery by adding trimethoprim to the cell culture medium. This means that, although scientists introduced the guide RNAs into the iPSCs, the genes targeted by guide RNAs were only activated or inhibited after iPSCs were differentiated into microglia-like cells.

If the expression of these targeted genes is disrupted, this could adversely impact the development of microglia. This could make it difficult to distinguish whether the change in expression of targeted genes impacted the development of microglia or the function of adult microglia.

This novel CRISPR platform thus enables scientists to assess gene function in adult microglia.

After validating the modified CRISPR screens, the researchers were able to identify genes in microglia involved in cellular processes such as proliferation, survival, activation of an immune response, and phagocytosis.

For instance, they identified genes that modulate phagocytosisthe cellular process of eliminating potentially toxic particles such as PFN1 and INPP5D, which have been implicated in neurodegenerative disorders.

Microglia respond adaptively to their local environment and exist in a wide range of context-specific states. Each microglial state, such as a diseased state, a healthy state, or the state while producing an immune response, is characterized by a specific gene expression profile.

The researchers used RNA sequencing at the single cell level to characterize different microglial states.

Based on the differences in gene expression profiles, the researchers were able to characterize nine distinct microglial states.

For instance, one of the functional states was characterized by the increased expression of the SPP1 gene that is upregulated in microglia in AD and other neurodegenerative conditions.

Moreover, by inhibiting the expression of genes using the CRISPR platform, the researchers were able to identify genes regulating the adoption of these functional states.

For instance, the researchers found that downregulating the colony-stimulating factor-1 receptor (CSF1R) gene using the CRISPR platform reduced the number of cells expressing high levels of the SPP1 gene.

Scientists observed a similar reduction in the number of microglia in the SPP1 diseased state upon using a drug that inhibits the CSF1R protein. Thus, by targeting genes or the proteins encoded by these genes that regulate the diseased state, scientists could switch microglia back to a healthy state.

Such findings show that this CRISPR-based platform could be used to identify the genes that regulate microglial states that are associated with neurodegenerative conditions. This could subsequently help scientists develop treatments that target these genes or the gene products.

CRISPR screens in human microglia have the potential to uncover therapeutic targets that can reprogram microglia to enhance their beneficial functions and block their toxicity in disease, explained the studys lead author, Dr. Martin Kampmann, a professor at the University of California, SF.

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Alzheimer's: Could controlling the brain's own clean-up crew help? - Medical News Today

Human iPSC co-culture model to investigate the interaction between microglia and motor neurons | Scientific Reports – Nature.com

Ethics statement

All human material (blood RNA, primary microglia RNA, iPSCs) used in this study was derived after signed informed consent: for blood, according to University of Oxford OHS policy document 1/03; all procedures related to the use of the primary microglia followed established institutional (McGill University, Montreal, QC, Canada) and Canadian Institutes of Health Research guidelines for the use of human cells; for iPSC, with approval from the South Central Berkshire Research Ethics Committee, U.K. (REC 10/H0505/71). The blood RNA and primary microglia RNA samples have been published previously26, as have the iPSC lines (see below).

Four healthy control iPSC lines, SFC840-03-03 (female, 67years old,35), SFC841-03-01 (male, 36,18), SFC856-03-04 (female, 78,36), OX3-06 (male, 49,37), generated from skin biopsy fibroblasts and characterized as described before, were used in this study. Additionally, the previously reported26 line AH016-3 Lenti_IP_RFP (male, 80years old), which constitutively expresses Red Fluorescent Protein (RFP) under continuous puromycin selection, was used for some live-imaging experiments.

iPSCs were cultured in mTeSR1 (StemCell Technologies) or OXE8 medium38 on Geltrex (Thermo Fisher)-coated tissue culture plates with daily medium changes. Passaging was done as clumps using EDTA in PBS (0.5mM). Cells were initially expanded at low passage to create a master stock, which was used for all experiments to ensure consistency. Cells were regularly tested negative for mycoplasma using MycoAlert Mycoplasma Detection Kit (Lonza).

iPSCs were differentiated to MNs according to our previously published protocol18,19,27. Briefly, neural induction of iPSC monolayers was performed using DMEM-F12/Neurobasal 50:50 medium supplemented with N2 (1X), B27 (1X), 2-Mercaptoethanol (1X), AntibioticAntimycotic (1X, all ThermoFisher), Ascorbic Acid (0.5M), Compound C (1M, both Merck), and Chir99021 (3M, R&D Systems). After two days in culture, Retinoic Acid (RA, 1M, Merck) and Smoothened Agonist (SAG, 500nM, R&D Systems) were additionally added to the medium. Two days later, Compound C and Chir99021 were removed from the medium. After another 5days in culture, neural precursors were dissociated using accutase (ThermoFisher), and split 1:3 onto Geltrex-coated tissue culture plates in medium supplemented with Y-27632 dihydrochloride (10M, R&D Systems). After one day, Y-27632 dihydrochloride was removed from the medium, and then the cells were cultured for another 8days with medium changes every other day. For terminal maturation, the cells were dissociated on day in vitro (DIV) 18 using accutase and plated onto coverslips or tissue culture plates coated with polyethylenimine (PEI, 0.07%, Merck) and Geltrex or tissue culture dishes coated with PDL (Sigma-Aldrich)/ Laminin (R&D Systems)/ Fibronectin (Corning). For this step, the medium was additionally supplemented with BDNF (10ng/mL), GDNF (10ng/mL), Laminin (0.5g/mL, all ThermoFisher), Y-27632 dihydrochloride (10M), and DAPT (10M, R&D Systems). Three days later, Y-27632 dihydrochloride was removed from the medium. After another three days, DAPT was removed from the medium. Full medium changes were then performed every three days.

For MNs differentiated in co-culture medium alone, all steps were performed similarly until three days after the terminal re-plating (D21). MNs were then cultured in co-culture medium as described below.

iPSCs were differentiated to macrophage/microglia precursors as described previously20,21. Briefly, embryoid body (EB) formation was induced by seeding iPSCs into Aggrewell 800 wells (STEMCELL Technologies) in OXE838 or mTeSR1 medium supplemented with Bone Morphogenetic Protein 4 (BMP4, 50ng/mL), Vascular Endothelial Growth Factor (VEGF, 50ng/mL, both Peprotech), and Stem Cell Factor (SCF, 20ng/mL, Miltenyi Biotec). After four days with daily medium changes, EBs were transferred to T175 flasks (~150 EBs each) and differentiated in X-VIVO15 (Lonza), supplemented with Interleukin-3 (IL-3, 25ng/mL, R&D Systems), Macrophage Colony-Stimulating Factor (M-CSF, 100ng/mL), GlutaMAX (1X, both ThermoFisher), and 2-Mercaptoethanol (1X). Fresh medium was added weekly. After approximately one month, precursors emerged into the supernatant and could be harvested weekly. Harvested cells were passed through a cell strainer (40M, Falcon) and either lysed directly for RNA extraction or differentiated to microglia in monoculture or co-culture as described below.

Three days after the final re-plating of differentiating MNs (DIV21), macrophage/microglia precursors were harvested as described above and resuspended in co-culture medium comprised of Advanced DMEM-F12 (ThermoFisher) supplemented with GlutaMAX (1X), N2 (1X), AntibioticAntimycotic (1X), 2-Mercaptoethanol (1X), Interleukin-34 (IL-34, 100ng/mL, Peprotech), BDNF (10ng/mL), GDNF (10ng/mL), and Laminin (0.5g/mL). MNs were quickly rinsed with PBS, and macrophage/microglia precursors re-suspended in co-culture medium were added to each well. Co-cultures were then maintained for at least 14days before assays were conducted as described below. Half medium changes were performed every 23days.

For comparisons between co-cultures and monocultures, MNs and monocultured microglia were also differentiated alone in co-culture medium.

Cells cultured on coverslips were pre-fixed with 2% paraformaldehyde in PBS for 2min and then fixed with 4% paraformaldehyde in PBS for 15min at room temperature (RT). After permeabilization and blocking with 5% donkey/goat serum and 0.2% Triton X-100 in PBS for 1h at RT, the coverslips were incubated with primary antibodies diluted in 1% donkey/goat serum and 0.1% Triton X-100 in PBS at 4C ON. The following primary antibodies were used: rabbit anti-cleaved caspase 3 (1:400, 9661S, Cell Signaling), mouse anti-ISLET1 (1:50, 40.2D6, Developmental Studies Hybridoma Bank), mouse anti-TUJ1 (1:500, 801201, BioLegend), rabbit anti-TUJ1 (1:500, 802001, BioLegend), chicken anti-TUJ1 (1:500, GTX85469, GeneTex), rabbit anti-IBA1 (1:500, 019-19741, FUJIFILM Wako Pure Chemical Corporation), goat anti-IBA1 (1:500, ab5076, abcam), rabbit anti-synaptophysin (1:200, ab14692, abcam), goat anti-ChAT (1:100, ab114P, abcam), rat anti-TREM2 (1:100, MAB17291-100, R&D Systems), rabbit anti-TMEM119 (1:100, ab185337, abcam), rat anti-CD11b (1:100, 101202, BioLegend).

After three washes with PBS-0.1% Triton X-100 for 5min each, coverslips were incubated with corresponding fluorescent secondary antibodies Alexa Fluor 488/568/647 donkey anti-mouse/rabbit/rat/goat, goat anti-chicken (all 1:1000, all ThermoFisher). Coverslips were then washed twice with PBS-0.1% Triton X-100 for 5min each and incubated with 4,6-diamidino-2-phenylindole (DAPI, 1g/mL, Sigma-Aldrich) in PBS for 10min. After an additional 5min-washing step with PBS-0.1% Triton X-100, the coverslips were mounted onto microscopy slides using ProLong Diamond Antifade Mountant (ThermoFisher). Confocal microscopy was then performed using an LSM 710 microscope (Zeiss).

For the analysis of neuronal and MN markers after differentiation, three z-stacks (2m intervals) of randomly selected visual fields (425.1425.1m) were taken for each coverslip at 20magnification. The ratios of TUJ1-positive, ChAT-positive, ISLET1-positive, ChAT-positive/ TUJ1-positive, and ISLET1-positive/ TUJ1-positive cells were then quantified using Fiji in a blinded fashion.

For the analysis of microglial markers in monoculture and co-culture, three z-stacks (1m intervals) of randomly selected visual fields (212.55212.55m) were taken for each coverslip at 40magnification. The expression of CD11b, TMEM119, and TREM2 in IBA1-positive cells in monoculture and co-culture was then quantified using Fiji.

For the analysis of apoptosis in neurons, five z-stacks images of randomly selected visual fields (212.55212.55m) were taken at 40magnification for each coverslip. The ratios of cleaved caspase 3/ TUJ1-positive cells were then quantified for neurons in monoculture and co-culture in a blinded fashion. For the analysis of apoptosis in microglia, three z-stacks images of randomly selected visual fields (212.55212.55m) were taken at 40magnification for each coverslip. The ratios of cleaved caspase 3/ IBA1-positive cells were then quantified for microglia in monoculture and co-culture.

For the analysis of microglial ramifications, five z-stacks images of randomly selected visual fields (212.55212.55m) were taken at 40magnification for each coverslip. To analyze the branching of IBA1-positive microglia in monoculture and co-culture, the average branch length, number of branch points and number of branch endpoints was determined using 3DMorph39, a Matlab-based script for the automated analysis of microglial morphology.

From the same harvest, macrophage precursors (pMacpre) were either lysed directly or differentiated to microglia in monoculture (pMGL) or microglia in co-culture with MNs (co-pMG) for 14days. pMGL were rinsed with PBS and directly lysed in the dish. For both pMacpre and pMGL, RNA was extracted using an RNAeasy Mini Plus kit (Qiagen) according to the manufacturers instructions. Co-cultures were first dissociated by 15min incubation with papain (P4762, Sigma-Aldrich) diluted in accutase (20 U/mL) and gentle trituration based on a previously published protocol40. The cell suspension was then passed through a cell strainer (70m, Falcon) to remove cell clumps. To extract co-pMG, magnetic-activated cell sorting (MACS) was then performed using CD11b-MACS beads (130093-634, Miltenyi Biotec) according to the manufacturers instructions. The panned cell population was lysed for RNA extraction using an RNAeasy Micro kit (Qiagen) according to the manufacturers instructions. In addition, RNA from human fetal microglia and blood monocytes from three different healthy genetic backgrounds wasre-used from our previous study26.

RNA from the four different healthy control lines (listed earlier) per condition (pMacpre, pMGL, co-pMG) was used for RNA sequencing analysis. Material was quantified using RiboGreen (Invitrogen) on the FLUOstar OPTIMA plate reader (BMG Labtech) and the size profile and integrity analysed on the 2200 or 4200 TapeStation (Agilent, RNA ScreenTape). RIN estimates for all samples were between 9.2 and 9.9. Input material was normalised to 100ng prior to library preparation. Polyadenylated transcript enrichment and strand specific library preparation was completed using NEBNext Ultra II mRNA kit (NEB) following manufacturers instructions. Libraries were amplified (14 cycles) on a Tetrad (Bio-Rad) using in-house unique dual indexing primers (based on41). Individual libraries were normalised using Qubit, and the size profile was analysed on the 2200 or 4200 TapeStation. Individual libraries were normalised and pooled together accordingly. The pooled library was diluted to~10nM for storage. The 10nM library was denatured and further diluted prior to loading on the sequencer. Paired end sequencing was performed using a NovaSeq6000 platform (Illumina, NovaSeq 6000 S2/S4 reagent kit, v1.5, 300 cycles), generating a raw read count of a minimum of 34M reads per sample.

Further processing of the raw data was then performed using an in-house pipeline. For comparison, the RNA sequencing data (GSE89189) fromAbud et al.28 and the dataset (GSE85839) fromMuffat et al.29 were downloaded and processed in parallel. Quality control of fastq files was performed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and MultiQC42. Paired-end reads were mapped to the human GRCh38.p13 reference genome (https://www.gencodegenes.org) using HISAT2 v2.2.143. Mapping quality control was done using SAMtools44 and Picard (http://broadinstitute.github.io/picard/) metrics. The counts table was obtained using FeatureCounts v2.0.145. Normalization of counts and differential expression analysis for the comparison of pMGL and co-pMG was performed using DESeq2 v1.28.146 in RStudio 1.4.1103, including the biological gender in the model and with the BenjaminiHochberg method for multiple testing correction. Exploratory data analysis was performed following variance-stabilizing transformation of the counts table, using heat maps and hierarchical clustering with the pheatmap 1.0.12 package (https://github.com/raivokolde/pheatmap) and principal component analysis. Log2 fold change (log2 fc) shrinkage for the comparison of pMGL and co-pMG was performed using the ashr package v2.2-4747. Genes with |log2 fc|>2 and adjusted p value<0.01 were defined as differentially expressed and interpreted with annotations from the Gene Ontology database using clusterProfiler v3.16.148 to perform over-representation analyses.

Equal amounts of RNA (30ng) were reverse-transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher) according to the manufacturers instructions. Quantitative real-time PCR was performed with Fast SYBR Green Master Mix (ThermoFisher) according to the manufacturers instructions using a LightCycler 480 PCR System (Roche). The following primers (ChAT from Eurofins Genomics, all others from ThermoFisher) were used:

Quantification of the relative fold gene expression of samples was performed using the 2Ct method with normalization to the GAPDH reference gene.

AH016-3 Lenti-IP-RFP-microglia were co-cultured with healthy control motor neurons in PEI- and Geltrex-coated glass bottom dishes for confocal microscopy (VWR). The RFP signal was used to identify microglia in co-culture. To visualize microglial movement, images of the RFP signal and brightfield were taken every~30s for 1h (22 stitched images, 20magnification) using a Cell Observer spinning disc confocal microscope (Zeiss) equipped with an incubation system (37C, 5% CO2). To image phagocytic activity, co-cultures were rinsed with Live Cell Imaging Solution (1X, ThermoFisher), and pHrodo Green Zymosan Bioparticles Conjugates (P35365, ThermoFisher) diluted in Live Cell Imaging Solution (50g/mL), which become fluorescent upon phagocytic uptake, were added. The dish was immediately transferred to the spinning disc confocal microscope, and stitched images (33, 20magnification) were acquired every 5min for 2h.

To induce pro-inflammatory (M1) or anti-inflammatory (M2) microglial phenotypes, cells were treated with Lipopolysaccharides (LPS, 100ng/mL, Sigma) and Interferon- (IFN-, 100ng/mL, ThermoFisher), or Interleukin-4 (IL-4, 40ng/mL, R&D Systems) and Interleukin-13 (IL-13, 20ng/mL, Peprotech), respectively, for 18h. Vehicle-treated (co-culture medium) cells were used as an unstimulated (M0) control.

To analyze the clustering of microglia upon pro-inflammatory and anti-inflammatory stimulation, RFP-positive microglia were imaged directly before the addition of M1/M2 inducing agents, and at 9h and 18h post-stimulation using the Cell Observer spinning disc confocal microscope (55 stitched images, 10magnification). The number of individual microglial cells and size of microglial clusters was quantified using the analyze particle function in Fiji.

After stimulation with M1/M2-inducing agents, culture supernatants were collected and spun down at 1200g for 10min at 4C. Pooled samples from three different healthy control lines for each cell type were analyzed using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems) according to the manufacturers instructions. The signal was visualized on a ChemiDoc MP imaging system (Bio-Rad) and analyzed using ImageStudioLite v5.2.5 (LI-COR). Data was then plotted as arbitrary units using the pheatmap 1.0.12 package in RStudio 1.4.1103.

In addition, to confirm the relative expression of Serpin E1 and CHI3L1 in cell culture supernatants, the Human Human Chitinase 3-like 1 Quantikine ELISA Kit (DC3L10) and Human Serpin E1/PAI-1 Quantikine ELISA Kit (DSE100, both R&D Systems) were used according to the manufacturers instructions.

pNeuron, pMGL and co-cultures were plated and maintained in WillCo-dish Glass Bottom Dishes (WillCo Wells) for 14days. Calcium transients were measured using the fluorescent probe Fluo 4-AM according to the manufacturers instructions (ThermoFisher). Cells were incubated with 20M Fluo 4-AM resuspended in 0.2% dimethyl sulfoxide for 30min at RT in Live Imaging Solution (ThermoFisher). After a washing step with Live Imaging Solution, cells were allowed to calibrate at RT for 1520min before imaging. Ca2+ images were taken by fluorescence microscopy at RT. The dye was excited at 488nm and images were taken continuously with a baseline recorded for 30s before stimulation. The stimuli used for calcium release were 50mM KCl (Sigma-Aldrich) for 30s, followed by a washing step for one minute. Microglial calcium release was stimulated by 50M ADP (Merck) under continuous perfusion for 1min, followed by a 1-min wash. Analysis of fluorescence intensity was performed using Fiji. Fluorescence measurements are expressed as a ratio (F/Fo) of the mean change in fluorescence (F) at a pixel relative to the resting fluorescence at that pixel before stimulation (Fo). The responses were analysed in 2040 cells per culture.

MNs on DIV 3345 were maintained in a bath temperature of 25C in a solution containing 167mM NaCl, 2.4mM KCl, 1mM MgCl2, 10mM glucose, 10mM HEPES, and 2mM CaCl2 adjusted to a pH of 7.4 and 300mOsm. Electrodes with tip resistances between 3 and 7M were produced from borosilicate glass (0.86mm inner diameter; 1.5mm outer diameter). The electrode was filled with intracellular solution containing 140mMK-Gluconate, 6mM NaCl, 1mM EGTA, 10mM HEPES, 4mM MgATP, 0.5mM Na3GTP, adjusted to pH 7.3 and 290mOsm. Data acquisition was performed using a Multiclamp 700B amplifier, digidata 1550A and clampEx 6 software (pCLAMP Software suite, Molecular Devices). Data was filtered at 2kHz and digitized at 10kHz. Series resistance (Rs) was continuously monitored and only recordings with stable<50 M and Rs<20% were included in the analysis. Voltage gated channel currents were measured on voltage clamp, neurons were pre-pulsed for 250ms with 140mV and subsequently a 10mV-step voltage was applied from 70 to+70mV. Induced action potentials were recorded on current clamp, neurons were held at 70mV and 8 voltage steps of 10mV, from 10 to 60mV, were applied. Data was analyzed using Clampfit 10.7 (pCLAMP Software suite).

Statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software, San Diego, California USA, http://www.graphpad.com). Comparisons of two groups were performed by two-tailed unpaired t-tests and multiple group comparisons by one-way or two-way analysis of variance (ANOVA) with appropriate post-hoc tests as indicated in the figure legends. The statistical test and number of independent experiments used for each analysis are indicated in each figure legend. Data are presented as single data points and meansSEM. Differences were considered significant when P<0.05 (*P<0.05; **P<0.01; ***P<0.001; ns: not significant). GraphPad Prism 9 or RStudio 1.4.1103 were used to plot data. Final assembly and preparation of all figures was done using Adobe Illustrator 25.4.1.

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Human iPSC co-culture model to investigate the interaction between microglia and motor neurons | Scientific Reports - Nature.com

What lab-grown cerebral organoids are revealing about the brain – New Scientist

Blobs of human brain cells cultivated in the lab, known as brain organoids or mini-brains, are transforming our understanding of neural development and disease. Now, researchers are working to make them more like the real thing

By Clare Wilson

Neil Webb

A DOZEN tiny, creamy balls are suspended in a dish of clear, pink liquid. Seen with the naked eye, they are amorphous blobs. But under a powerful microscope, and with some clever staining, their internal complexity is revealed: intricate whorls and layers of red, blue and green.

These are human brain cells, complete with branching outgrowths that have connected with one other, sparking electrical impulses. This is the stuff that thoughts are made of. And yet, these collections of cells were made in a laboratory in this case, in the lab of Madeline Lancaster at the University of Cambridge.

The structures, known as brain organoids or sometimes mini-brains, hold immense promise for helping us understand the brain. They have already produced fresh insights into how this most mysterious organ functions, how it differs in people with autism and how it goes awry in conditions such as dementia and motor neurone disease. They have even been made to grow primitive eyes.

To truly fulfill the potential of mini-brains, however, neuroscientists want to make them bigger and more complex. Some are attempting to grow them with blood vessels. Others are fusing two organoids, each mimicking a different part of the brain. Should they succeed, their lab-grown brains could model development and disease in the real thing in greater detail than ever before, paving the way to new insights and treatments.

But as researchers seek to make mini-brains genuinely worthy of the name, they move ever closer to a crucial question: at what point will their creations approach sentience?

The key to developing organoids was the discovery of stem cells,

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What lab-grown cerebral organoids are revealing about the brain - New Scientist

Cell Culture Media Market Size Worth $10.2 Billion by 2030: Grand View Research, Inc. – PR Newswire

SAN FRANCISCO, July 21, 2022 /PRNewswire/ -- The global cell culture media market size is expected to reach USD 10.2 billion by 2030, according to a new report by Grand View Research, Inc. The market is expected to expand at a CAGR of 12.1% from 2022 to 2030. Expansion of biosimilars and biologics, growth in stem cell research, and emerging bio manufacturing technologies for cell-based vaccines are the major factors which are likely to drive the market. For instance, in October 2021, the Australian Government funded the Australian-led stem cell research through USD 25 million in grants.

Key Industry Insights & Findings from the report:

Read 150-page market research report, "Cell Culture Media Market Size, Share & Trends Analysis Report By Product (Serum-free Media, Classical Media), By Type (Liquid Media, Semi-solid And Solid Media), By Application, By End-user, By Region, And Segment Forecasts, 2022 - 2030", published by Grand View Research.

Cell Culture Media Market Growth & Trends

The expansion of clear, regulatory approval paths for biosimilars in emerging markets is generating great opportunities for biosimilar monoclonal antibodies. The availability of an approval pathway in the U.S., has led to new opportunities for bio manufacturers to enter major markets around the globe. Biosimilar versions of monoclonal antibodies have the probability to offer cost reductions of 25-30%, and many emerging market countries are vigorously developing pathways for approvals and are swiftly catching up. As this industry is expanding the key biopharmaceutical players are adopting robust culturing technologies to meet the increasing demand; thereby driving the growth of the market.

Moreover, there is growing interest in improving the stem cell culture, because this technology is being used extensively in research for studying the stem cell biology, as well as for therapeutic applications. Furthermore, funding related to this research field has augmented in recent years which has accelerated the growth of the market. In addition to this, key media manufacturers launched new products for stem cell research. For instance, in September 2021, Bio-Techne Corporation launched a novel medium for the maintenance and expansion of induced pluripotent stem cells having applications in both translational and research workflows.

The outbreak of COVID-19 pandemic has improved the demand for well-established cell-based vaccine production technologies. Moreover, it has given rise to a few scientific innovations, particularly in the production and testing of vaccine technology. For instance, the Vero line originated from the African green monkey kidney and has been extensively used for viral vaccine manufacturing. It has also been used for the development of various SARS-CoV variants. ProVeroTM1 Serum-free Medium is one such medium manufactured by Lonza Bioscience which is protein-free, and of non-animal origin designed to support the growth of Vero cells and MDCK.

Moreover, in many European countries, cell-based flu vaccines have been approved. A probable advantage of cell culture technology is that it authorizes faster start-up of the manufacturing of vaccines during the pandemic. Today, the development of superior biological models, the optimization of culture growth medium, and the reduced dependence on animal-derived components endure to drive the rapidly developing vaccine development.

On the other hand, ethical issues concerning the use of animal-derived products hinders the industry growth. For instance, FBS is collected from the blood of fetal calves is one of the major ethical issues of serum containing media. It is projected that 600,000 liters of FBS is achieved from up to 1.8 million bovine fetuses are produced globally every year, presenting momentous scientific and ethical challenges. To overcome this issue, numerous workshops were held in the past on the replacement of fetal bovine serum and possible ways to reduce the use of FBS in media.

Cell Culture Media Market Segmentation

Grand View Research has segmented the global cell culture media market based on product, application, type, end-user, and region:

Cell Culture MediaMarket - Product Outlook (Revenue, USD Million, 2018 - 2030)

Cell Culture MediaMarket - Application Outlook (Revenue, USD Million, 2018 - 2030)

Cell Culture MediaMarket - Type Outlook (Revenue, USD Million, 2018 - 2030)

Cell Culture MediaMarket - End-user Outlook (Revenue, USD Million, 2018 - 2030)

Cell Culture MediaMarket - Regional Scope Outlook (Revenue, USD Million, 2018 - 2030)

List of Key Players of Cell Culture Media Market

Check out more related studies published by Grand View Research:

Browse through Grand View Research's Biotechnology Industry Research Reports.

About Grand View Research

Grand View Research, U.S.-based market research and consulting company, provides syndicated as well as customized research reports and consulting services. Registered in California and headquartered in San Francisco, the company comprises over 425 analysts and consultants, adding more than 1200 market research reports to its vast database each year. These reports offer in-depth analysis on 46 industries across 25 major countries worldwide. With the help of an interactive market intelligence platform, Grand View Research Helps Fortune 500 companies and renowned academic institutes understand the global and regional business environment and gauge the opportunities that lie ahead.

Contact:

Sherry James Corporate Sales Specialist, USA Grand View Research, Inc. Phone: +1-415-349-0058 Toll Free: 1-888-202-9519 Email: [emailprotected] Web: https://www.grandviewresearch.com Grand View Compass| Astra ESG Solutions Follow Us: LinkedIn | Twitter

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Cell Culture Media Market Size Worth $10.2 Billion by 2030: Grand View Research, Inc. - PR Newswire

Dean Kamen on the power of celebrating your own obsoletion – TechCrunch

More than 40 years and 1,000 or so patents after selling his first company, AutoSyringe, to healthcare giant Baxter, Dean Kamen still gets a charge describing breakthrough innovation. Its been five years since his organ fabricating project ARMI (Advanced Regenerative Manufacturing Institute) divided critics.

The project made more waves early last month, at the CNN-hosted conference Life Itself. Kamen paints the picture appearing on a panel at TC Sessions: Robotics today:

Doris Taylor, who moved up here from where she spent more than a decade in Texas, at the Texas Heart Institute, she gets on stage with a beaker. In the beaker is a miniature, pediatric-scale beating heart that was manufactured with induced pluripotent stem cells were put into a scaffold of preexisting organ. Within an hour of that presentation, Martine Rothblatt, the founder and chairman of United Therapeutics, is on stage and they roll out from backstage an almost surrealistic, lit from the top of the box. A panel opens, and what emerges out of the top of this platform is a scaffold of a human lung, that was printed, entirely printed at the smallest scale any printer has ever operated.

Inventor Dean Kamen looks on as over 110,000 pounds of personal protective equipment (PPE), shipped from Shanghai, China, is unloaded from a cargo plane at Manchester-Boston Regional Airport in Manchester, New Hampshire, Thursday, April 30, 2020. The equipment will be used for medical workers and first responders in their fight against the virus outbreak. (AP Photo/Charles Krupa)

Kamen is first to admit, however, that the path to all success is paved with failure. The trick is learning the right lesson.

What Ive learned from failure is go back and decide was the fundamental goal wrong thats why it failed, you succeeded, but nobody needs this or did the available technology and your systems integration and application have it wrong, in which case, youve now learned enough, go try again, go use a different approach, Kamen explains. Pick yourself up, try again, using a different approach. And it really doesnt matter how many times you fall down. If you fall down five times, but you stand up six, its okay. And in the end, you only need a win every once in a while to keep your confidence up. And hopefully, to give you the resources to keep going even though inevitably youll have failures, let the projects fail, dont let the people fail.

These are among the fundamentals Kamen has attempted to infuse into FIRST, the education program he co-founded in 1989, with MIT professor Woodie Flowers. It is best known for its robotics competitions, which center around competitive builds of robots and other projects, bringing the teamwork and enthusiasm of sports to STEM education subjects that might otherwise turn off students who traditionally encounter them in more formal and staid settings.

Kids wont go to class, or theyll take math for 45 minutes between phonics and spelling, one day a week. But theyll go after school for three hourse, every single day to get better at football or get better at basketball. So I said, look, were not competing for the hearts and minds of kids with the science fair and the spelling bee, were competing with the things that they invest all of their time, energy and passion in. So lets use that model make it aspirational, make it after school. Dont give them quizzes and tests, give them letters and trophies. Bring the school band and the mascots.

U.S. Sen. Jeanne Shaheen (D-NH), right, looks toward inventor Dean Kamen as over 110,000 pounds of personal protective equipment (PPE) from Shanghai, China, delivered to protect medical workers and first responders fighting the COVID-19 virus outbreak, is unloaded from a cargo plane at Manchester-Boston Regional Airport in Manchester, New Hampshire, Thursday, April 30, 2020. (AP Photo/Charles Krupa)

Perhaps the hardest-fought lesson of all, however, is understanding, accepting and even welcoming the fact that progress in technology and sciences means that one day your best work will be eclipsed.

You have to be more than prepared for it. You have to be confident it will happen, and you have to celebrate it. I celebrate it more when its me that obsoleted the last thing I did, but if somebody else can obsolete it and if I get to a point where I need a better clinical solution than a dialysis machine or an insulin pump, if I can get to a place with somebody elses technology to gave me a new organ or a prosthetic limb or something, I need to have a better quality of life, I will thank that person. And I hope I will return that favor by giving them something of value that we invented.

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Dean Kamen on the power of celebrating your own obsoletion - TechCrunch

CRISPR Technology Highlights Genes That Contribute to the Development of Emphysema and COPD – Boston Medical Center

BOSTON Researchers from the Center for Regenerative Medicine at Boston Medical Center and Boston University School of Medicine used variants of CRISPR to understand the functions of the genes that cause emphysema and chronic obstructive pulmonary disease (COPD). Published in Science Advances, researchers discovered functional consequences by turning off the expression of the gene that contributes to the pathogenesis of these diseases.

This is the first time that CRISPRi and CRISPRa have been applied in human induced pluripotent stem cells to understand the functional role of these genes, says Andrew Wilson, MD, a pulmonologist at Boston Medical Center and associate professor of medicine at Boston University School of Medicine. It gets us closer to understanding how inherited factors help contribute to susceptibility to emphysema.

COPD and emphysema is the third leading cause of death worldwide, creating a significant burden of disease. Emphysema is a complex genetic disease caused by a mutation or variant in a number of genes that contribute to making some individuals more susceptible to disease than others. Genome-wide association studies (GWAS) have implicated variants in or near a growing number of genes, but understanding their functions and how they potentially contribute to the development of COPD and emphysema is quite limited.

There have been no new significant pharmacological agents developed to help treat the large number of patients afflicted with COPD or emphysema worldwide, says Rhiannon Werder, MD, a postdoctoral fellow at the Center for Regenerative Medicine at Boston Medical Center and Boston University School of Medicine. Our hope is that this study will help in the understanding of the genetics of the disease, improve our understanding of how the disease occurs at a cellular level, and support the development of new therapies to treat these conditions.

Researchers devised a system using variants of CRISPR to either turn off expression of a gene of interest using CRISPR interference (CRISPRi) or overexpress a gene of interest using CRISPR activation (CRISPRa) in induced pluripotent stem cells (iPSCs). Researchers grew these cells in a dish and differentiated them to generate cells that reside in the lung. The cell type studied is called the type 2 alveolar epithelial cell, a progenitor cell for the alveolus the alveolus is the part of the lung where gas exchange occurs and is the structure that is damaged in emphysema. So by understanding how GWAS genes affect type 2 cells, researchers can start to understand how these might contribute to diseases that affect these cells, like emphysema.

Once type 2 cells were generated, researchers then used CRISPRi to turn off expression of nine different GWAS genes and analyzed them to see how the cells were affected, especially their ability to proliferate, something that they need to be able to do in response to injury like that which occurs in emphysema. Researchers noticed that turning off one particular gene, desmoplakin (DSP), caused the cells to increase their proliferation and increased their expression of genes associated with cellular maturation. Researchers found that cells in which DSP expression was turned off before smoke exposure turned off expression of cell junction genes to a greater degree than in controls. These were also better at forming new colonies, a measure of progenitor function, than controls. Researchers then looked in mice that had DSP deleted from their lung epithelial cells, compared to control mice with normal DSP. Researchers found that the type 2 cells in the DSP deletion mice were more proliferative following injury, consistent with findings in human iPSC-derived type 2 cells.

DSP appears to modulate the proliferative capacity of type 2 cells at baseline and following injuries that are relevant to human disease, such as smoke exposure. Lower levels of DSP expression increase the proliferative capacity of type 2 cells in the system, potentially making them better able to respond to an injury. In contrast, higher levels of expression as found in cells containing the variant associated with COPD risk by GWAS appear to make the cells less proliferative after smoke exposure, potentially explaining how this gene contributes to disease.

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CRISPR Technology Highlights Genes That Contribute to the Development of Emphysema and COPD - Boston Medical Center

The ‘Benjamin Button’ effect: Scientists can reverse aging in mice. The goal is to do the same for humans – KITV Honolulu

In molecular biologist David Sinclair's lab at Harvard Medical School, old mice are growing young again.

Using proteins that can turn an adult cell into a stem cell, Sinclair and his team have reset aging cells in mice to earlier versions of themselves. In his team's first breakthrough, published in late 2020, old mice with poor eyesight and damaged retinas could suddenly see again, with vision that at times rivaled their offspring's.

"It's a permanent reset, as far as we can tell, and we think it may be a universal process that could be applied across the body to reset our age," said Sinclair, who has spent the last 20 years studying ways to reverse the ravages of time.

"If we reverse aging, these diseases should not happen. We have the technology today to be able to go into your hundreds without worrying about getting cancer in your 70s, heart disease in your 80s and Alzheimer's in your 90s." Sinclair told an audience at Life Itself, a health and wellness event presented in partnership with CNN.

"This is the world that is coming. It's literally a question of when and for most of us, it's going to happen in our lifetimes," Sinclair told the audience.

"His research shows you can change aging to make lives younger for longer. Now he wants to change the world and make aging a disease," said Whitney Casey, an investor who is partnering with Sinclair to create a do-it-yourself biological age test.

While modern medicine addresses sickness, it doesn't address the underlying cause, "which for most diseases, is aging itself," Sinclair said. "We know that when we reverse the age of an organ like the brain in a mouse, the diseases of aging then go away. Memory comes back; there is no more dementia.

"I believe that in the future, delaying and reversing aging will be the best way to treat the diseases that plague most of us."

In Sinclair's lab, two mice sit side by side. One is the picture of youth, the other gray and feeble. Yet they are brother and sister, born from the same litter -- only one has been genetically altered to age faster.

If that could be done, Sinclair asked his team, could the reverse be accomplished as well? Japanese biomedical researcher Dr. Shinya Yamanaka had already reprogrammed human adult skin cells to behave like embryonic or pluripotent stem cells, capable of developing into any cell in the body. The 2007 discovery won the scientist a Nobel Prize, and his "induced pluripotent stem cells," soon became known as "Yamanaka factors."

However, adult cells fully switched back to stem cells via Yamanaka factors lose their identity. They forget they are blood, heart and skin cells, making them perfect for rebirth as "cell du jour," but lousy at rejuvenation. You don't want Brad Pitt in "The Curious Case of Benjamin Button" to become a baby all at once; you want him to age backward while still remembering who he is.

Labs around the world jumped on the problem. A study published in 2016 by researchers at the Salk Institute for Biological Studies in La Jolla, California, showed signs of aging could be expunged in genetically aged mice, exposed for a short time to four main Yamanaka factors, without erasing the cells' identity.

But there was a downside in all this research: In certain situations, the altered mice developed cancerous tumors.

Looking for a safer alternative, Sinclair lab geneticist Yuancheng Lu chose three of the four factors and genetically added them to a harmless virus. The virus was designed to deliver the rejuvenating Yamanaka factors to damaged retinal ganglion cells at the back of an aged mouse's eye. After injecting the virus into the eye, the pluripotent genes were then switched on by feeding the mouse an antibiotic.

"The antibiotic is just a tool. It could be any chemical really, just a way to be sure the three genes are switched on," Sinclair said. "Normally they are only on in very young developing embryos and then turn off as we age."

Amazingly, damaged neurons in the eyes of mice injected with the three cells rejuvenated, even growing new axons, or projections from the eye into the brain. Since that original study, Sinclair said his lab has reversed aging in the muscles and brains of mice and is now working on rejuvenating a mouse's entire body.

"Somehow the cells know the body can reset itself, and they still know which genes should be on when they were young," Sinclair said. "We think we're tapping into an ancient regeneration system that some animals use -- when you cut the limb off a salamander, it regrows the limb. The tail of a fish will grow back; a finger of a mouse will grow back."

That discovery indicates there is a "backup copy" of youthfulness information stored in the body, he added.

"I call it the information theory of aging," he said. "It's a loss of information that drives aging cells to forget how to function, to forget what type of cell they are. And now we can tap into a reset switch that restores the cell's ability to read the genome correctly again, as if it was young."

While the changes have lasted for months in mice, renewed cells don't freeze in time and never age (like, say, vampires or superheroes), Sinclair said. "It's as permanent as aging is. It's a reset, and then we see the mice age out again, so then we just repeat the process.

"We believe we have found the master control switch, a way to rewind the clock," he added. "The body will then wake up, remember how to behave, remember how to regenerate and will be young again, even if you're already old and have an illness."

Studies on whether the genetic intervention that revitalized mice will do the same for people are in early stages, Sinclair said. It will be years before human trials are finished, analyzed and, if safe and successful, scaled to the mass needed for a federal stamp of approval.

While we wait for science to determine if we too can reset our genes, there are many other ways to slow the aging process and reset our biological clocks, Sinclair said.

"The top tips are simply: Focus on plants for food, eat less often, get sufficient sleep, lose your breath for 10 minutes three times a week by exercising to maintain your muscle mass, don't sweat the small stuff and have a good social group," Sinclair said.

What controls the epigenome? Human behavior and one's environment play a key role. Let's say you were born with a genetic predisposition for heart disease and diabetes. But because you exercised, ate a plant-focused diet, slept well and managed your stress during most of your life, it's possible those genes would never be activated. That, experts say, is how we can take some of our genetic fate into our own hands.

Cutting back on food -- without inducing malnutrition -- has been a scientifically known way to lengthen life for nearly a century. Studies on worms, crabs, snails, fruit flies and rodents have found restricting calories "delay the onset of age-related disorders" such as cancer, heart disease and diabetes, according to the National Institute on Aging. Some studies have also found extensions in life span: In a 1986 study, mice fed only a third of a typical day's calories lived to 53 months -- a mouse kept as a pet may live to about 24 months.

Studies in people, however, have been less enlightening, partly because many have focused on weight loss instead of longevity. For Sinclair, however, cutting back on meals was a significant factor in resetting his personal clock: Recent tests show he has a biological age of 42 in a body born 53 years ago.

"I've been doing a biological test for 10 years now, and I've been getting steadily younger for the last decade," Sinclair said. "The biggest change in my biological clock occurred when I ate less often -- I only eat one meal a day now. That made the biggest difference to my biochemistry."

Sinclair incorporates other tools into his life, based on research from his lab and others. In his book "Lifespan: Why We Age and Why We Don't Have To," he writes that little of what he does has undergone the sort of "rigorous long-term clinical testing" needed to have a "complete understanding of the wide range of potential outcomes." In fact, he added, "I have no idea if this is even the right thing for me to be doing."

With that caveat, Sinclair is willing to share his tips: He keeps his starches and sugars to a minimum and gave up desserts at age 40 (although he does admit to stealing a taste on occasion). He eats a good amount of plants, avoids eating other mammals and keeps his body weight at the low end of optimal.

He exercises by taking a lot of steps each day, walks upstairs instead of taking an elevator and visits the gym with his son to lift weights and jog before taking a sauna and a dip in an ice-cold pool. "I've got my 20-year-old body back," he said with a smile.

Speaking of cold, science has long thought lower temperatures increased longevity in many species, but whether it is true or not may come down to one's genome, according to a 2018 study. Regardless, it appears cold can increase brown fat in humans, which is the type of fat bears use to stay warm during hibernation. Brown fat has been shown to improve metabolism and combat obesity.

Sinclair takes vitamins D and K2 and baby aspirin daily, along with supplements that have shown promise in extending longevity in yeast, mice and human cells in test tubes.

One supplement he takes after discovering its benefits is 1 gram of resveratrol, the antioxidant-like substance found in the skin of grapes, blueberries, raspberries, mulberries and peanuts.

He also takes 1 gram of metformin, a staple in the arsenal of drugs used to lower blood sugars in people with diabetes. He added it after studies showed it might reduce inflammation, oxidative damage and cellular senescence, in which cells are damaged but refuse to die, remaining in the body as a type of malfunctioning "zombie cell."

However, some scientists quibble about the use of metformin, pointing to rare cases of lactic acid buildup and a lack of knowledge on how it functions in the body.

Sinclair also takes 1 gram of NMN, or nicotinamide mononucleotide, which in the body turns into NAD+, or nicotinamide adenine dinucleotide. A coenzyme that exists in all living cells, NAD+ plays a central role in the body's biological processes, such as regulating cellular energy, increasing insulin sensitivity and reversing mitochondrial dysfunction.

When the body ages, NAD+ levels significantly decrease, dropping by middle age to about half the levels of youth, contributing to age-related metabolic diseases and neurodegenerative disorders. Numerous studies have shown restoring NAD+ levels safely improves overall health and increases life span in yeast, mice and dogs. Clinical trials testing the molecule in humans have been underway for three years, Sinclair said.

"These supplements, and the lifestyle that I am doing, is designed to turn on our defenses against aging," he said. "Now, if you do that, you don't necessarily turn back the clock. These are just things that slow down epigenetic damage and these other horrible hallmarks of aging.

"But the real advance, in my view, was the ability to just tell the body, 'Forget all that. Just be young again,' by just flipping a switch. Now I'm not saying that we're going to all be 20 years old again," Sinclair said.

"But I'm optimistic that we can duplicate this very fundamental process that exists in everything from a bat to a sheep to a whale to a human. We've done it in a mouse. There's no reason I can think of why it shouldn't work in a person, too."

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The 'Benjamin Button' effect: Scientists can reverse aging in mice. The goal is to do the same for humans - KITV Honolulu