Stem Cell Clinic

To better understand Parkinson’s disease, this San Diego expert sent her own cells to space – The San Diego Union-Tribune

Jeanne Loring likes to say shes been to space without her feet even leaving the ground.

Just weeks ago, the Scripps Research Institute professor of molecular medicine sent some of her own genetically mapped cells to space as part of first-of-its-kind research to study the progression and onset of Parkinsons disease, multiple sclerosis and other neurodegenerative diseases.

I love traveling. Ive been on all the continents, and so I figured, whats left? Loring said jokingly. I just jumped at the opportunity when I learned that it was possible.

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In July, the cells arrived via cargo spacecraft at the International Space Station, where they remained under close observation for about a month 250 miles above Earth, and traveling at 17,500 miles per hour before they splashed back down to Earth last week.

The study is part of new National Stem Cell Foundation-funded neurodegeneration research to observe how cells communicate in microgravity in a way not possible on Earth, explained Paula Grisanti, founder and CEO of the foundation.

Its really pure exploration at this point, because theres no history of anybody doing this before, she said. Were paving the path.

An organoid derived from Dr. Jeanne Lorings induced pluripotent stem cells is prepared to be sent to the International Space Station.

(Courtesy of Dr. Davide Marotta)

Loring, a Del Mar resident who is one of the worlds leading experts in Parkinsons and a senior scientific advisor for the foundation, has been working with human-induced pluripotent stem cells since the technology was first discovered in 2006.

Called organoids, these cells are made from human skin tissue, which is put into a culture dish and turned into pluripotent stem cells, Loring explained.

Pluripotent stem cells only exist in culture dishes, they dont exist in our bodies, she said. Pluripotent means they can form any cell type in the body so for Loring, that meant forming nerve cells to create brain-like structures.

Its hard to study peoples brains, Loring said. You can do all this external stuff like they do with physical exams, but theres not any window into the brain so this is providing a sort of brain avatar.

Organoids provide a stand-in for the brain that can be studied by researchers, Loring explained. They make connections with each other, the cells talk to each other, so in a lot of ways, its a really good model of the brain, she added.

Moreover, the organoids mimic the brains of people with MS and Parkinsons.

Loring has been working with these organoids for years through Aspen Neuroscience, a San Diego-based company she co-founded that is working to create the worlds first personalized cell therapy for Parkinsons, using a patients own cells so they dont have to worry about rejection. Clinical trials may start as early as next year, she said.

Tubes containing neural organoids are loaded into a rack in preparation for placement in Cube Lab to travel to the International Space Station.

(Courtesy of Space Tango)

For the last four years, the foundations team of bicoastal researchers has been working together to study these organoids in space.

While an experiment in space presents its own challenges, Loring said its worth the work, as researchers hope to gain valuable and unique insight into how disorders like Parkinsons and MS develop. You can see them interacting and talking to each other in 3-D in a way that you cannot on Earth, Grisanti said.

Along with Lorings healthy organoids, which are used as a control, organoids derived from patients with Parkinsons and MS were sent to the space station, while the entire experiment was replicated on Earth.

Specifically, researchers are studying the neuroinflammation in the organoids, which is like when the immune system in the brain is overactive, Grisanti explained.

Organoid cultures are sealed in holders and ready to be placed in Cube Lab for space flight. The cover shows National Stem Cell Foundations SpaceX CRS-25 mission patch.

(Courtesy of Space Tango)

What we hope to find is a point at which things start to go wrong in those neurodegenerative diseases, where we could then intervene with a new drug or cell therapy, she said. And were seeing signs that that happens more in space than it does on the ground, so it helps create the type of interaction that you would see early in a neurodegenerative disease.

Grisanti said they hope to be able to use this research to develop a new drug or cell therapy to treat these disorders and potentially other neurodegenerative diseases in the future.

I think weve cracked the door open, but weve got some more flying to do, she added.

Read more here:
To better understand Parkinson's disease, this San Diego expert sent her own cells to space - The San Diego Union-Tribune

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 -...

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

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

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