Fort Worth Biotech Innovator Honored With Lifetime Achievement … – dallasinnovates.com

Stella Robertson [Image: Courtesy photo, iamguru/istockphoto, DI]

Longtime Fort Worth biotech innovator and investor Stella Robertson has been honored with a 2023 ARVO Foundation award recognizing her lifetime of work in vision research and philanthropy.

Robertsons expertise and guidance has helped countless entrepreneurs and start-ups, making her a true inspiration in biotech and the field of ophthalmology. The award was given in recognition for her long-standing support of the Women in Eye and Vision Research (WEAVR) initiative and her generous philanthropic support of the foundation.

The ARVO Foundation is the philanthropic arm of ARVO (The Association of Research in Vision and Ophthalmology). WEAVR is the Foundation initiative supporting women in vision research.

Robertson and Suchi Acharya, founder and CEO of Ayuvis Research in Fort Worth, were speakers at ARVOs B2B education course this year. The course, Bench to Bedside, is a translational research and pitch workshop for ARVO members.

I have always wanted my research to make a difference in peoples lives, to solve problems and help them have a better life, Robertson said in an Arvo Q&A in 2019.

The scientist is known for her work at Fort Worth-based Alcon where she launched roughly 17 products, and is co-founder at Bios Partners, a venture capital firm focusing on life sciences based in the city. Bios Partners was founded in 2015 by Robertson, along with managing partners Aaron G.L. Fletcher and Les Kreis.

With over 25 years of experience in pharmaceutical research and development, she has a wealth of knowledge to share.

During her time at Alcon, where she was vice president in R&D at Alcon Laboratories, Inc., a division of Novartis, she grew and led organizations responsible for the ophthalmic pipeline, including pharmaceuticals and medical devices. Robertson developed some of the first human ocular cell lines used for drug discovery and successfully launched sixteen ophthalmic medications to treat ocular allergy, pain, inflammation, glaucoma, uveitis, and infection.

Her research interests are diverse, ranging from local immune and inflammatory mechanisms to diagnostics and drug delivery. Robertson is a published author and also holds several patents.

The scientist received a Ph.D. in biology-immunology from Johns Hopkins University, was an Arthritis Foundation postdoctoral research fellow at UTHSC Dallas, and completed the Program for Management Development at Harvard Business School.

Today, as the founder of Arrochar Consulting, Robertson specializes in due diligence, translational research, product development and life sciences, providing support to entrepreneurs and start ups in emerging technology.

Robertsons passion for helping others extends beyond her consulting work. She volunteers and mentors with TECH Fort Worth, a local non-profit incubator/accelerator, and local university entrepreneurial program. She also serves as a corporate board member, board observer, and scientific advisor for early-stage companies. In addition, Robertson is a member and investor with Cowtown Angels, an angel investment network based in Fort Worth.

My focus now is on giving back, according to the ARVO Q&A. Her dedication to the field is apparent through her involvement in various organizations such as Women in Ophthalmology and ARVO (IM section). Shes served on multiple committees and sits on the ARVO Foundation Board.

Robertson is credited with expanding the research and entrepreneurial community in Fort Worth and encouraging students to stay in STEM education. Her advice to young women scientists is to get the best training in their chosen field, find something that makes them feel fulfilled, and persevere through many nos in their careers, she said in the Q&A.

A career in research is a life choice, Robertson has advised. But, she adds, dont forget to take time for yourself and family. Together you will delight in and discover the world, nature, and research again through their eyes.

Quincy Preston contributed to this report.

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Slated to be built in Fort Worth's Historic Southside neighborhood, the planned $70 million museum will get the city funding once the balance for the project has been raised. Designed by the New York office of Denmark-based Bjarke Ingels Group, the building will house the museum on its second level, with a business incubator, restaurant, 250-seat amphitheater, and storefronts at ground level. Literally and figuratively, it was designed to be a beacon of light in an area that has been dark for a very long time, says Jarred Howard, principal of the project's developer.

Entrepreneurs and industry leaders benefit from the city's business-friendly approach.

North Texas has plenty to see, hear, and watch.Here are our editors' picks. Plus, you'll find more selections to "save the date."

You'll find deadlines coming up for a new accelerator program; and many more opportunities.

Rhithm, a Dallas social-emotional learning and mental health startup, raised $4 million in a seed round last year for its emoji-based bio-social assessments app, which is now used by over 2,400 schools in 29 states, according to the company. One district that adopted the app is Fort Worth ISDand it recently announced a change in how the app will be used.

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Single-Use Bioreactors Market is Expected to Reach $10.0 billion … – GlobeNewswire

Chicago, April 28, 2023 (GLOBE NEWSWIRE) -- The single-use bioreactor industry is expected to experience tremendous growth in the near future due to increased demand for more cost-effective and efficient production of biopharmaceuticals. The use of single-use bioreactors has the potential to reduce capital investment and operational costs, as well as increase production flexibility. This is expected to lead to a significant increase in the number of companies investing in and utilizing single-use bioreactors. Additionally, ongoing research and development of new technologies and systems, such as advanced sensors and advanced analytics, are expected to further drive growth in the near future. Finally, the growing global demand for biopharmaceuticals is expected to increase the demand for single-use bioreactors, further driving growth in the industry.

Single-Use Bioreactors market in terms of revenue was estimated to be worth $4.2 billion in 2023 and is poised to reach $10.0 billion by 2028, growing at a CAGR of 19.0% from 2023 to 2028 according to a latest report published by MarketsandMarkets. The factors driving the growth of this market include the increasing adoption of single-use bioreactors among startups and SMEs, lower operational complexity of single-use bioreactors compared to conventional stainless-steel bioreactors, reduced energy and water consumption, growing size of the biologics and biosimilars market, and technologically advanced offerings by players in single-use bioreactors. However, extractability and leachability issues regarding disposable of single-use components used in bioreactors and regulatory concerns related to single-use bioreactors are the major factors restraining the growth of this market to certain extent.

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Single-Use Bioreactors Market Scope:

Based on product, the single-use bioreactors market is segmented into single-use bioreactor systems, single-use media bags, single-use filtration assemblies, and other products. The single-use bioreactor systems segment dominated the single-use bioreactors market in 2022. Single-use bioreactor systems offer advantages such as low capital investment, low operating expenses, and lower environmental footprint.

Based on type, the single-use bioreactors market is segmented into stirred-tank single-use bioreactors, wave-induced single-use bioreactors, bubble-column single-use bioreactors, and other single-use bioreactors such as hybrid bioreactors and single-use bioreactors with vertically perforated discs. The stirred-tank single-use bioreactors segment dominated the market in 2022 owing to stirred-tank single-use bioreactors in the culturing of aerobic microbial cell cultures.

Based on the type of molecule, the global single-use bioreactors market is segmented into monoclonal antibodies (mAbs), vaccines, gene-modified cells, stem cells, and other molecules. In 2022, the monoclonal antibodies segment accounted for the largest share of the global single-use bioreactors market owing to the increasing demand for single-use bioreactors in the manufacturing of mAbs, owing to low investment costs and a reduction in time-intensive changeover procedures.

Based on the type of cell, the global single-use bioreactors market is segmented into mammalian cells, bacterial cells, yeast cells, and other cells (insect and plant cells). In 2022, the mammalian cells segment accounted for the largest share of the market. The increasing adoption of mammalian cells due to their post-translational modification capacity and molecular structure assembly that closely resembles proteins in humans are the major factors driving the growth of this segment.Based on application, the single-use bioreactors market is segmented into research & development, process development, and bioproduction. The bioproduction segment accounted for the largest share of the market in 2022 and is projected to register the highest CAGR during the forecast period owing to the increasing use of single-use bioreactors in biomanufacturing and the increasing demand for single-use bioreactors in CMOs due to the advantages it offers, such as flexibility and easy scalability.

Based on end users, the single-use bioreactors market is segmented into pharmaceutical & biotechnology companies, CROs & CMOs, and academic & research institutes. The pharmaceutical & biotechnology companies segment accounted for the largest share of the single-use bioreactors market in 2022 owing to the increasing R&D initiatives by pharmaceutical, biopharmaceutical, and biotechnology companies and growing production of biologics & biosimilars.

Based on region, single-use bioreactors market is segmented into North America, Europe, Asia Pacific, Latin America, and the Middle East & Africa. In 2022, Asia Pacific has the fastest growth rate owing to The factors such as the growing biopharmaceutical industry, growing investments by pharmaceutical & biotechnology companies in the Asia Pacific region, and the growing number of CROs & CMOs in different countries in the region are supporting the growth of the market in the region.

Key Market Players:

Major players operating in the single-use bioreactors market include Sartorius AG (Germany), Thermo Fisher Scientific (US), Danaher Corporation (US), and Merck Millipore (Germany). These companies have manufacturing units as well as strong distribution networks across key regions, such as North America, Europe, Asia Pacific, Latin America and the Middle East & Africa. They have an established portfolio of reputable services, a robust market presence, and strong business strategies. Furthermore, these companies have a significant market share, and vast service portfolio.

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Hypothetic Challenges of Single-Use Bioreactors Market in Near Future:

Top 3 Use Cases of Single-Use Bioreactors Market:

Recent Developments:

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Appendix 4C Quarterly Activity ReportMesoblast Financial and … – BioSpace

NEW YORK, April 27, 2023 (GLOBE NEWSWIRE) -- Mesoblast Limited (Nasdaq:MESO; ASX:MSB), global leader in allogeneic cellular medicines for inflammatory diseases, today provided an activity report for the third quarter ended March 31, 2023.

Financial Highlights

Operational Highlights

Remestemcel-L

Rexlemestrocel-L

OtherSalary payments to full-time Executive Directors were US$330,756 and fees to Non-Executive Directors were US$197,365, detailed in Item 6 of the Appendix 4C cash flow report for the quarter.4

A copy of the Appendix 4C Quarterly Cash Flow Report for the third quarter FY2023 is available on the investor page of the companys website http://www.mesoblast.com.

About Mesoblast Mesoblast is a world leader in developing allogeneic (off-the-shelf) cellular medicines for the treatment of severe and life-threatening inflammatory conditions. The Company has leveraged its proprietary mesenchymal lineage cell therapy technology platform to establish a broad portfolio of late-stage product candidates which respond to severe inflammation by releasing anti-inflammatory factors that counter and modulate multiple effector arms of the immune system, resulting in significant reduction of the damaging inflammatory process.

Mesoblast has a strong and extensive global intellectual property portfolio with protection extending through to at least 2041 in all major markets. The Companys proprietary manufacturing processes yield industrial-scale, cryopreserved, off-the-shelf, cellular medicines. These cell therapies, with defined pharmaceutical release criteria, are planned to be readily available to patients worldwide.

Mesoblast is developing product candidates for distinct indications based on its remestemcel-L and rexlemestrocel-L allogeneic stromal cell technology platforms. Remestemcel-L is being developed for inflammatory diseases in children and adults including steroid refractory acute graft versus host disease, biologic-resistant inflammatory bowel disease, and acute respiratory distress syndrome. Rexlemestrocel-L is in development for advanced chronic heart failure and chronic low back pain. Two products have been commercialized in Japan and Europe by Mesoblasts licensees, and the Company has established commercial partnerships in Europe and China for certain Phase 3 assets.

Mesoblast has locations in Australia, the United States and Singapore and is listed on the Australian Securities Exchange (MSB) and on the Nasdaq (MESO). For more information, please see http://www.mesoblast.com, LinkedIn: Mesoblast Limited and Twitter: @Mesoblast

References / Footnotes

Forward-Looking StatementsThis press release includes forward-looking statements that relate to future events or our future financial performance and involve known and unknown risks, uncertainties and other factors that may cause our actual results, levels of activity, performance or achievements to differ materially from any future results, levels of activity, performance or achievements expressed or implied by these forward-looking statements. We make such forward-looking statements pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995 and other federal securities laws. Forward-looking statements should not be read as a guarantee of future performance or results, and actual results may differ from the results anticipated in these forward-looking statements, and the differences may be material and adverse. Forward-looking statements include, but are not limited to, statements about: the initiation, timing, progress and results of Mesoblasts preclinical and clinical studies, and Mesoblasts research and development programs; Mesoblasts ability to advance product candidates into, enroll and successfully complete, clinical studies, including multi-national clinical trials; Mesoblasts ability to advance its manufacturing capabilities; the timing or likelihood of regulatory filings and approvals, manufacturing activities and product marketing activities, if any; the commercialization of Mesoblasts product candidates, if approved; regulatory or public perceptions and market acceptance surrounding the use of stem-cell based therapies; the potential for Mesoblasts product candidates, if any are approved, to be withdrawn from the market due to patient adverse events or deaths; the potential benefits of strategic collaboration agreements and Mesoblasts ability to enter into and maintain established strategic collaborations; Mesoblasts ability to establish and maintain intellectual property on its product candidates and Mesoblasts ability to successfully defend these in cases of alleged infringement; the scope of protection Mesoblast is able to establish and maintain for intellectual property rights covering its product candidates and technology; estimates of Mesoblasts expenses, future revenues, capital requirements and its needs for additional financing; Mesoblasts financial performance; developments relating to Mesoblasts competitors and industry; and the pricing and reimbursement of Mesoblasts product candidates, if approved. You should read this press release together with our risk factors, in our most recently filed reports with the SEC or on our website. Uncertainties and risks that may cause Mesoblasts actual results, performance or achievements to be materially different from those which may be expressed or implied by such statements, and accordingly, you should not place undue reliance on these forward-looking statements. We do not undertake any obligations to publicly update or revise any forward-looking statements, whether as a result of new information, future developments or otherwise.

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Appendix 4C Quarterly Activity ReportMesoblast Financial and ... - BioSpace

Cell and Tissue Preservation Market To Deliver Greater Revenues … – Digital Journal

PRESS RELEASE

Published April 28, 2023

Polaris Market Research endeavors to present the most relevant and admirable research on Cell and Tissue Preservation Market: By Size, Trends, Share, Growth, Segments, Industry Analysis and Forecast, 2032 based on the needs of the business. The report examines the constant changes taking place in the market, and such dynamics are helping the industry in growing its operations. The primary goal of this report is to highlight the growing potential of the market and its growth-promoting variables. The report also examines the Cell and Tissue Preservation Market growth rate and valuation. The study provides a thorough analysis of current industry trends and developments, as well as a complete predictive and prescriptive analysis.

According to the research report, the global cell and tissue preservation market was valued at USD 3.47 billion in 2021 and is expected to reach USD 8.32 billion by 2030, to grow at a CAGR of 10.5% during the forecast period.

The cell and tissue preservation market refers to the industry that develops and provides products and services for the storage, transport, and preservation of biological samples such as cells, tissues, and organs. The preservation of these samples is essential for research, drug development, and clinical applications.

The report determines the upcoming trends and the competitive landscape of the industry that helps companies make insightful decisions and manage business growth effectively. It contains history analysis, key developments in the market, projected growth, geographical analysis, Cell and Tissue Preservation Market share, revenue, industry variable, key segmentation, and forecast scenario. Segmentation is prominently performed by type, application, players, and region. Furthermore, the regional and country-level section assesses the market in each geography and Cell and Tissue Preservation Market size by region and country.

The market for cell and tissue preservation has been growing in recent years due to the increasing demand for biobanking, regenerative medicine, and personalized medicine. Biobanking refers to the collection and storage of biological samples for future research, while regenerative medicine aims to develop new treatments by using cells and tissues to repair or replace damaged tissues or organs.

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The Following Are the Main Advantages of This Market Research

Competitive Landscape Overview

Cell and Tissue Preservation Market key players are examined with sizeable market shares, revenue, business strategies, recent advancements, and growth rates. Recent activities for these businesses, including the introduction of fresh products or services, research projects, geographic expansions, and technological developments, are taken into account when determining their standing in this market. The most recent developments of major market participants, including their capacities, plant turnarounds, expansions, investments, mergers, and acquisitions, are also covered in the research.

Top Key Players:

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A Brief About Geography

In this section, the analysts have investigated prospective regions that can bring manufacturers success in the upcoming years. The geographical study provides accurate volume and value of Cell and Tissue Preservation Market forecasts, assisting participants in gaining a comprehensive understanding of the entire sector. Every region has been profiled in terms of basis point share, year-over-year growth forecasts, and significant laws that apply to that region. The country-level and local-level analysis has also been included in order to promote high-rise growth while discouraging growth inhibitors and lowering market limitations.

Regions Covered in This Report Are

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Furthermore, to capture each detail of the market, it is essential to understand the market dynamics deeply. Thus, readers are advised to go through the market dynamics section, which includes an analysis of key factors and their impact on the market, drivers from both the supply as well as demand side, and restraints that are expected to impede the industry growth. Also, a thorough analysis of the industry accompanied by graphs, pie charts, and numbers makes it simple to understand.

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Bold new therapy delivery method shows initial promise as treatment for Duchenne muscular dystrophy – Medical Xpress

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Graphical abstract. Credit: Cell (2023). DOI: 10.1016/j.cell.2023.03.033

Doug Millay, Ph.D., a scientist with the Division of Molecular Cardiovascular Biology at Cincinnati Children's has dedicated his career to revealing the most fundamental mechanisms of skeletal muscle development. He has been a leader in characterizing how two "fusogens" called Myomaker and Myomerger mediate the entry of stem cells into mature muscle cells to build the tissue that humans depend upon for movement, breathing, and survival.

Now, some of the basic discoveries made by Millay and colleagues are translating into a potential treatment for people living with Duchenne muscular dystrophy (DMD). Their latest research, published April 12, 2023, in the journal Cell, reveals that in mice, modified viruses, engineered with Myomaker and Myomerger, result in specific fusion with muscle cells. These viruses can therefore be used as a vector to deliver a vital gene needed for muscle function that is mutated in people with DMD.

A key unknown prior to this work was whether proteins like Myomaker and Myomerger, which mainly function on cells, could even work on viruses. First author Sajedah Hindi, Ph.D., also with the Division of Molecular Cardiovascular Biology at Cincinnati Children's and a leading member of the research team, took on the challenge to test this idea.

Hindi first designed a strategy to place Myomaker and Myomerger on the surface of viruses and showed that they were functional in cultured cells. She went on to leverage her extensive experience in skeletal muscle biology to test the efficacy of these novel vectors in mice.

"This modified viral vector appears to be a promising tool for delivering a potential lifelong supply of the gene that is absent in people with DMD," Millay says. "The unique advantages of this vector provide an opportunity to significantly impact gene therapy for a myriad of muscle diseases."

DMD is a rare and fatal genetic muscle disease characterized by the lack of a critical membrane-stabilizing protein called dystrophin, which results in progressive muscle degeneration and weakness. DMD primarily strikes boys, occurring in about 1 of every 3,500 male births worldwide.

Doctors often diagnose the disease between ages 3 and 6 when children show early signs of significant muscle weakness, such as delayed ability to sit, stand, or walk and difficulties learning to speak. Over time, DMD becomes fatal as muscle degeneration disrupts lung and heart function.

There is no cure. However, lifespans have been extended and quality of life has been improved for many through physical therapy and medications to address certain symptoms. Some gene therapy clinical trials are evaluating the use of adeno-associated virus (AAV) as the delivery vector, and there is hope that these strategies work. However, novel vectors, such as the lentiviruses described by Millay and colleagues, have the potential to improve long-term delivery of therapeutic material for muscular dystrophies.

Conducting the numerous experiments involved in this study took Hindi and collaborators about four years to complete. A significant collaborator who helped initiate the project was Benjamin Podbilewicz, from the Technion-Israel Institute of Technology, Haifa, Israel. Their findings include:

Much more research will be needed to further develop this discovery into a treatment that could someday benefit people with DMD. Even more work will be needed to determine which other muscle diseases might be treated with this lentivirus vector.

"We envision that this concept, transferring a naturally occurring process within muscle to membrane vehicles, could revolutionize delivery of therapeutic material to skeletal muscle to improve genetic conditions such as muscular dystrophy and conditions associated with muscle loss and weakness," Millay says.

More information: Sajedah M. Hindi et al, Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery, Cell (2023). DOI: 10.1016/j.cell.2023.03.033

Journal information: Cell

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Bold new therapy delivery method shows initial promise as treatment for Duchenne muscular dystrophy - Medical Xpress

Regulation of synaptic connectivity in schizophrenia spectrum by … – Nature.com

The methods were performed in accordance with relevant guidelines and regulations and approved by the Ethics Committee of the University Hospital and Faculty of Medicine Tuebingen. We confirm that participants provided a written informed consent to take part in the study. Inclusion and exclusion criteria for the selection of patients diagnosed with SCZ are described in Supplementary Table1. iPSCs were generated and fully characterized as described elsewhere (Table1; refs. 22,23). All experiments were carried out with all lines in parallel.

Patient-derived human fibroblasts were reprogrammed by nucleofection of non-integrative, episomal vectors encoding for OCT3/4, SOX2, LIN28, KLF4, c-MYC, p53 and EBNA1 (Addgene, catalog no. 41813, 41814, 41855, 41856, 41857). Electroporated fibroblasts were seeded onto Matrigel (Corning, catalog no. 354277) coated well plates and expanded in a feeder-free culture system for 2128 days until first iPS colonies appeared. iPS clones were manually picked and expanded on Matrigel in mTeSR Plus medium (STEMCELL Technologies, catalog no. 05825). Passaging was routinely performed non-enzymatically using Gentle Cell Dissociation Reagent (STEMCELL Technologies, catalog no. 100-0485) in early passage numbers or later enzymatically by using accutase (Sigma Aldrich, catalog no. A6964). All expanded iPS clones were routinely tested for expression of stem cell marker on protein and RNA level and pluripotency. All iPS clones used in this study were chromosomally intact.

For microglia differentiation, we modified a previously published protocol for the differentiation of iPSC into monocytes and macrophages55. iPSCs were dissociated using accutase and seeded at a density of 5104 cells per cm (day -2). Only wells containing equally distributed iPSC colonies of 1020 cells were considered for differentiation. For all differentiation steps, a 1:1 mix of IMDM without phenol red (Thermo Fisher Scientific, catalog no. 21056023) and Hams F12 Nutrient Mix (Thermo Fisher Scientific, catalog no. 21765029) was used. The basal medium was supplemented with 10g/mL poly vinyl alcohol (Sigma Aldrich, catalog no. P8136), 64g/mL ascorbic acid 2 phosphate (Sigma Aldrich, catalog no. A8960), 0.1x chemically defined lipid concentrate (Thermo Fisher Scientific, catalog no. 11905031), 2x ITS-X (Thermo Fisher Scientific, catalog no. 51500056), 0.0039% -Monothioglycerol (Sigma Aldrich, catalog no. M6145), 1x GlutaMAX (Thermo Fisher Scientific, catalog no. 35050061) and 1x non-essential amino acids. Additional growth factors and cytokines were always added freshly before usage. For mesoderm induction at day 0, the basal medium was supplemented with 50ng/mL BMP4 (Peprotech, catalog no. 120-05ET), 15ng/mL Activin A (Miltenyi Biotec, catalog no. 130-115-008) and 1.5M CHIR99021 (Axon Medchem, catalog no. 1386) for mesoderm induction. For the suppression of self-renewal in favor of stem cell differentiation at day 2, 10M of SB431542 and SCF (Peprotech, catalog no. 300-07), VEGF (Peprotech, catalog no. 100-20) and bFGF (Bio-Techne, catalog no. 233-FB) were added to the medium at a final concentration of 50ng/ml each. For hematopoietic patterning at day 5, 10ng/ml of IL-3 (Peprotech, catalog no. 200-03) and 50ng/ml of IL-6 (Peprotech, catalog no. 200-06), 50ng/ml of TPO (Miltenyi Biotec, catalog no. 130-095-747), 50ng/ml of bFGF, 50ng/ml of SCF and 50ng/ml of VEGF were supplemented to the basal medium. Medium was refreshed at day 7. At day 9, differentiated cells grew to full confluence with hematopoietic stem cells emerging into the supernatant. Adherent cells were dissociated by accutase treatment and added to non-adherent cells collected from the supernatant. After centrifugation at 300xg for 3min, cells were resuspended in microglia medium containing 100ng/ml of IL-34 (Peprotech, catalog no. 200-34), 50ng/ml of TGF-1 (Peprotech, catalog no. 100-21) and 25ng/ml of GM-CSF (Peprotech, catalog no. 300-03). Cells were subsequently plated on ultra-low attachment plates that were pretreated with Anti-Adherence Rinsing Solution for at least 5min and afterwards rinsed twice with DPBS. Microglia differentiation was allowed to proceed for further seven days with medium changes every other day.

Microglia were routinely characterized regarding expression of key markers like IBA1, SPI1 and TMEM119. Functionality was proven by active uptake of pHrodo-labelled bacteria and response to LPS as a pro-inflammatory stimulus. Microglia identity was confirmed by RNA sequencing. Transcriptome analysis and bioinformatical evaluation was performed by CeGaT GmbH (Germany) as previously described15. For characterization of microglial phenotypes, microglial genes were chosen according to previously published literature that identified panels of highly specific microglia signature genes21,56,57,58,59,60.

Secretion of the pro-inflammatory cytokine TNF was quantified by a standard sandwich-ELISA (human TNF-alpha DuoSet ELISA kit, R&D Systems, catalog no. DY210) according to the manufacturers instructions. Briefly, 96-well plates were coated with the capture antibody and incubated over night at room temperature. Wells were washed three times and blocked for at least 1h at room temperature. Wells were again washed and 100l of culture supernatant or standards were added and incubated for 2h at room temperature. After washing, detection antibody was added and incubated at room temperature for 2h. Wells were washed and the streptavidin / horse radish peroxidase (HRP) mix was added for 30min at room temperature. Afterwards, wells were washed again and substrate solution was added for 20min at room temperature in the dark, stop solution was added and the plate was tapped for mixing. Immediately afterwards, the optical density was determined using a microplate reader (Tecan Spark) set to 450nm with wavelength corrections set to 540nm.

Flow cytometry measurements were performed using BD FACS Chorus software on a BD FACS Melody and analyzed using FlowJo 10.6.1 (FlowJo Engine, Becton Dickinson & Company). Cells were detached, washed three times with DPBS and stained with conjugated antibodies for 30min at 4C. Subsequently, cells were washed three times with DPBS and resuspended in PBS+1% FCS for immediate analysis. The following conjugated antibodies were used: anti-human SSEA-4 PE-Vio770 (Miltenyi Biotech, catalog no. 130-105-081), anti-human CD11b FITC (Thermo Fisher Scientific, catalog no. 11-0118-42) and anti-human CD45 VioBlue (Miltenyi Biotech, catalog no. 130-110-775). Doublets were excluded in FSC and SSC. Unstained cells served as negative population.

Day 19 microglia were plated at a density of 1105 cells/cm on Matrigel-coated 96 well plates. Cells adhered within 24h and were subsequently fixed and stained against NFB p65 (Cell Signaling, catalog no. 6956T), Phalloidin CruzFluor 488 Conjugate (Santa Cruz Biotechnology, catalog no. sc-363791) and Hoechst (Sigma-Aldrich, catalog no. 911004450). Using confocal laser scan microscopy with a 63x plan-apochromatic oil immersion objective, at least ten 3D Z-stacks were acquired of microglia were taken within each experiment. During acquisition, all settings such as exposure time, laser intensity and gain were kept constant. Z-stacks were further processed using Imaris software (Bitplane, version 8.2.0). Therefore, a surface for the nucleus was generated covering the Hoechst signal. Within this mask, the mean fluorescence intensity of NFB p65 was determined and quantified.

Caspase-1 activity was determined using the Caspase-Glo 1 Inflammasome Assays (Promega, catalog no. G9951) according to the manufacturers instructions. Briefly, day 19 microglia were plated at a density of 1.2105 cells/cm on Matrigel-coated 96 well plates and incubated overnight. The next day, cells were treated with 100ng/ml of LPS (Sigma Aldrich, catalog no. L6529) for 3h and subsequently with 5mM ATP (Sigma Aldrich, catalog no. A2383) for 30min at 37C and 5% CO2. The culture supernatant was transferred into a white 96 well plate and Caspase-Glo 1 Reagent was added. The mixture was incubated at room temperature in the dark for 1h and luminescence was measured on a Tecan Spark microplate reader.

Day 19 microglia and nave iPSC as control were plated at a density of 2104 cells per well of a 96 well plate and cells adhered within 24h. pHrodo Red E. coli BioParticles (Thermo Fisher Scientific, catalog no. P35361) were resuspended in 2ml PBS to generate a stock suspension with a concentration of 1mg/ml. Bioparticles were vortexed rigorously to generate a homogenous suspension. 10l of pHrodo Red E. coli BioParticles were added to the wells. Cells were incubated at 37C and 5% CO2 for 4h in the Incucyte S3 live-cell imaging system (Sartorius). 9 images per well were acquired every 15min at x20 magnification. Finally, the relative red fluorescent units per image were analyzed over time.

For lentivirus production, HEK293FT were cultured at 37C and 8 % CO2 in culture medium consisting of DMEM (Thermo Fisher Scientific, catalog no. 10566016), 10% FCS (Thermo Fisher Scientific, catalog no. 10270106), 500g/ml G418 (Carl Roth, catalog no. 2039), 1% non-essential amino acids (Thermo Fisher Scientific, catalog no. 11140035) and passaged using 0.25% Trypsin/EDTA (Thermo Fisher Scientific, catalog no. 25200056) once or twice a week. For lentivirus production, cells were dissociated and seeded at a density of 3000 cells per cm. After four days of incubation, medium of HEK293FT cells was changed to a serum-reduced transfection medium of Opti-MEM (Thermo Fisher, catalog no. 11058021) supplemented with 5% FCS. 27g of pC-Pack2 Lentiviral Packaging Mix (Cellecta, catalog no. CPCP-K2A) were mixed with 108l of Lipofectamine 2000 Reagent (Thermo Fisher Scientific, catalog no. 11668019) in 4.5mL Opti-MEM, incubated at room temperature for 20min and added to the cells for further incubation at 37C and 5% CO2. After 24h, medium was changed, while after 48h and 72h post-transfection the supernatant was removed and stored at 80C. Lentiviral suspensions were filtered through a 22nm filter, transferred into ultracentrifugation buckets and centrifuged at 19,600rpm and 4C for 80min. Pellets were air dried for a few minutes and remaining liquid was removed with sterilized soft tissue papers. Finally, 100l of DPBS+1% BSA were added per tube without pipetting or resuspending. Tubes were sealed with Parafilm and left overnight at 4C. The next day, pellets were resuspended by pipetting several times and aliquoted for storage at 80C. Titer determination was performed using the Lenti-X p24 Rapid Titer Kit (Takara Bio, catalog no. 632200) according to the manufacturers instructions. Lentiviral suspensions were diluted 10-fold and 100-fold and quantified against a p24 standard curve. Yields ranged from 51010 to 51011 particles/ml.

Ectodermal patterning was induced using the STEMdiff Neural Induction Kit (STEMCELL Technologies, catalog no. 05835) according to the manufacturers instructions. iPSC were dissociated using accutase and 2106 iPSC were seeded into ultra-low attachment AggreWell 800 well plates (STEMCELL Technologies, catalog no. 34815) pretreated with Anti-Adherence Rinsing Solution (STEMCELL Technologies, catalog no. 07010). After cultivation at 37C and 5% CO2 for seven days with daily medium changes, embryoid bodies were harvested using 37m reversible strainers (STEMCELL Technologies, catalog no. 27215). Prior to seeding, 6-well plates were pretreated with 20% poly-L-ornithine (PLO, Sigma-Aldrich, catalog no. P4957) in Dulbeccos phosphate-buffered saline (Thermo Fisher Scientific, catalog no. 14190094), incubated for 2h at room temperature and washed three times with DMEM/F12 (Thermo Fisher Scientific, catalog. no. 21331020). Subsequently, wells were treated with 10g/ml laminin (Lam, Sigma-Aldrich, catalog no. L2020) diluted in DMEM/F12 and incubated overnight at 37C and 5% CO2. Harvested embryoid bodies were washed to remove remaining single cells and seeded onto PLO/Lam pre-coated well plates in STEMdiff Neural Induction Medium with daily medium changes. Neural rosettes were selected using the STEMdiff Neural Rosette Selection Reagent (STEMCELL Technologies, catalog no. 05832), resuspended in STEMdiff Neural Induction Medium supplemented with 1M Dorsomorphin dihydrochloride (Bio-Techne, catalog no. 3093), 10M SB 431542 (Bio-Techne, catalog no. 1614), 500ng/ml recombinant Human Noggin Fc Chimera Protein (Bio-Techne, catalog no. 719-NG) and cultivated in PLO/Lam coated 6-well plate. After the first passage, cultivation medium was changed to STEMdiff Neural Progenitor Medium (STEMCELL Technologies, catalog no. 05833). NPCs were passaged up to passage 10. All generated NPCs were routinely tested for progenitor marker expression, such as PAX6, NESTIN or SOX1.

Neuronal differentiation was achieved by lentiviral overexpression of human Neurogenin 2 following previously published protocols61,62. 3.15104 NPC were dissociated by accutase treatment and seeded in PLO/Lam-coated well plates at a density of 3104 cells per cm in STEMdiff Neural Progenitor Medium. For induction of neuronal differentiation62, NPC were co-infected with lentiviral vectors pLV-TetO-hNGN2-Puro (Addgene, catalog no. 79049), and FUdeltaGW-rtTA (Addgene, catalog no. 19780) at a final concentration of approximately 10ng/ml or 2108 particles/ml per lentivirus. After 24h, doxycycline (Sigma Aldrich, catalog no. D9891) was added to a final concentration of 10g/ml to induce tetracycline-dependent expression of the reverse tetracycline transactivator (rtTA) and hNGN2. 24h later, 2g/ml of puromycine (Thermo Fisher Scientific, catalog no. 11113803) was added to the medium to select for transduced NPC. After removal of selection medium at day 2 post transduction, cells were supplied with neuronal differentiation medium consisting of Neurobasal Plus Medium (Thermo Fisher Scientific, catalog no. A3582901) supplemented with 1x B27 Plus supplement (Thermo Fisher Scientific, catalog no. A3582801), 1x N2 supplement (Thermo Fisher Scientific, catalog no. 17502048), 1g/ml Laminin, 20ng/mL BDNF (Peprotech, catalog no. 450-02), 20ng/mL GDNF (Peprotech, catalog no. 450-10), 500g/mL dibutyryl cyclic adenosine monophosphate (Sigma Aldrich, catalog no. D0627), 35g/mL L-Ascorbic Acid (Sigma Aldrich, catalog no. A2078) and 10g/ml doxycycline. At this point, 3104 murine primary astrocytes per cm were added a 50% medium change was performed every other day until neurons were assayed or fixed after 1421 days in vitro.

Neuronal and microglial differentiation started separately from each other for 16 days. Subsequently, microglia were lifted from the ultra-low attachment plates, washed with DPBS, centrifuged and finally resuspended in microglia medium. In case of pretreatment, microglia were primed using 100ng/ml of LPS or 10M of Minocycline (STEMCELL Technologies, catalog no. 74112) at 37C and 5% CO2 for 60min. Subsequently, microglial cells were washed with DPBS and added to the neuronal cultures. For a final microglia:neuron ratio of approximately 1:5, microglia were seeded at a density of 5104 microglial cells per cm combined with 3104 initially seeded NPCs per cm. The co-culture plate was transferred to the incubator and left for 72h at 37C and 5% CO2. Co-cultures were maintained in microglia medium throughout the experiments.

iPSC-derived neurons and microglia, cultured in 96-well clear plates (Greiner Bio, catalog no. 655090), were fixed using paraformaldehyde (4% in PBS, Sigma Aldrich, catalog no. P6148) for 15min at room temperature. After fixation, cells were washed three times with PBS and then blocked and permeabilized at room temperature in 0.1% Triton X-100/PBS containing 1X Blocking Reagent for ELISA (Merck, catalog no. 11112589001) for 30min. After overnight incubation at 4C with primary antibodies diluted in blocking solution, cells were washed three times in PBS and exposed to fluorescently labeled secondary antibodies (1:500; Cy3 anti-rabbit (Jackson ImmunoResearch, catalog no. 111-165-144) or Cy5-coupled goat anti-mouse secondary antibodies (Jackson ImmunoResearch, catalog no. 115-175-146) and Alexa Fluor 488-coupled goat anti-chicken or 647-coupled goat anti-rat antibodies (Thermo Fisher Scientific, catalog no. A21247, A11039). Secondary antibodies were dissolved in blocking solution and incubated at room temperature for 2h. Nuclei were stained using Hoechst Dye 33258 (1:1,000 in PBS, Sigma-Aldrich, catalog no. 911004450). The following primary antibodies were used: mouse monoclonal anti-Beta-Tubulin III (STEMCELL Technologies, catalog no. 60100, 1:250), mouse monoclonal CX3CR1 (BioLegend, catalog no. 355701, 1:500), rabbit polyclonal anti-IBA1 (FUJIFILM Wako Chemicals, catalog no. 019-19741, 1:1000), CD11b monoclonal antibody (ICRF44), eBioscience (#14-0118-82), rat monoclonal anti-LAMP1 (Santa Cruz Biotechnology, catalog no. sc-19992, 1:100), chicken polyclonal anti-MAP2 (Invitrogen, catalog no. PA1-10005; 1:2500), mouse monoclonal anti-NFB p65 (Cell Signaling, catalog no. 6956), mouse monoclonal anti-PAX6 (BioLegend, catalog no. 862001, 1:200), Phalloidin CruzFluor# 488 (Santa Cruz Biotechnology, catalog no. sc-363791), rabbit monoclonal recombinant anti-PSD95 (Synaptic Systems, catalog no. 124008, 1:500), mouse monoclonal anti-SPI1 (PU.1, BioLegend, catalog no. 658002, 1:100), rabbit polyclonal anti-SOX1 (Abcam, catalog no. ab22572, 1:500), mouse monoclonal anti-Synapsin1 (Synaptic Systems, catalog no. 106011, 1:1000), rabbit polyclonal anti-Synaptophysin1 (Synaptic Systems, catalog no. 101002, 1:500), rabbit anti-TMEM119 (Synaptic Systems, catalog no. 400002, 1:400), rabbit monoclonal anti-TREM2 (Cell Signaling, catalog no. 91068, 1:400), mouse monoclonal anti-VGlut1 (Synaptic Systems, catalog no. 135511, 1:300), mouse monoclonal anti-Nestin (Synaptic System, catalog no. 312011, 1:1000). Antibody specificity was confirmed by analysis on differentiated cells and nave iPSC, and by secondary antibody only stainings.

To determine microglial pruning of synaptic structures, Z-stacks of neuronal networks were acquired with a confocal laser scan microscopy Cell Observer SD with a x63 plan-apochromatic oil immersion objective. Z-stacks were retrieved from regions of comparable fibre density, while the settings for acquisition (such as exposure time, laser intensity and gain) were unchanged for all conditions. Each image is a 3D reconstruction of a z-stack.

Images of neuronal cultures or neuron-microglia co-cultures were further processed by imaging using Imaris software. A surface was generated covering all MAP2 signals present in the whole stack. Next, the surface was masked using the Synapsin 1 (SYN1) signal creating a new channel for SYN1. After spot detection in the new SYN1 channel, SYN1-positive synaptic structures were counted after thresholding and referred to the volume of MAP2-positive structures to provide the density of SYN1-positive presynaptic terminals. The threshold for SYN1 spot detection was kept constant for each replicate. Data from multiple images were averaged to give yield to one datapoint for each biological replicate. The number of biological replicates is indicated in the figure legends. Within each image 2-3 microglial cells were analyzed on average. Within individual biological replicates, samples were normalized to the mean of CTR1.

Microglial uptake of synaptic structures was quantified by determination of the mean fluorescence intensity of SYN1 within IBA1 positive microglia. To this end, Z-stacks of microglia were acquired as described above and further processed using Imaris. A first surface was generated using the IBA1 signal to cover whole microglial cells and was subsequently masked with the signal for SYN1. Mean fluorescence intensities were measured for SYN1-positive spots identified within microglia. At least three independent experiments were performed for each donor combination.

Statistical analysis was performed using GraphPad Prism 9.2.0 (GraphPad Software Inc.). For non-Gaussian distribution in pairwise comparisons, the unpaired MannWhitney U test was performed and for group comparisons, KruskalWallis test with Dunns post-hoc multiple comparisons test was used. The type of statistical tests used and results are reported in the figure legends or main text.

Further information on research design is available in theNature Portfolio Reporting Summary linked to this article.

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Parse Biosciences and Human Cell Atlas Partner to Help Map … – PR Web

The HCA has already provided transformational work to the single cell community. Parses technology will assist this important work with unprecedented scale, opening up new possibilities for what is achievable in scientific research.

SEATTLE (PRWEB) April 27, 2023

Parse Biosciences, a leading provider of accessible and scalable single cell sequencing solutions, today announced the company has partnered with the international Human Cell Atlas (HCA) consortium, a global collaboration of researchers developing comprehensive reference maps of all human cells as a basis for understanding human health and diagnosing, monitoring, and treating disease.

Parses EvercodeTM technology for single cell transcriptomics and single cell immune profiling enables million-cell experiments without the need for expensive instrumentation. Through the partnership, HCA members gain more access to Parses technology. Parse will also provide support to HCA members for experimental design, assay execution, and bioinformatic support in applying the Evercode technology to their research.

The Human Cell Atlas project is an enormous undertaking and will transform our understanding of the 37 trillion cells in the human body, noted Dr Sarah Teichmann, co-Chair of the HCA Organizing Committee and Head of Cellular Genetics at Englands Wellcome Sanger Institute. Our global community of researchers is charting the cell types in the body, across time from development to adulthood and eventually to old age, and effective large-scale technologies are needed to enable this.

An open global initiative, the HCA was founded in 2016 and has grown to more than 2,900 members from over 1,500 institutes and 94 countries around the world. Bringing together an international community of biologists, clinicians, technologists, physicists, computational scientists, software engineers and mathematicians, HCA membership is open to the entire scientific community worldwide.

The HCA has already provided transformational work to the single cell community. Parses technology will assist this important work with unprecedented scale, opening up new possibilities for what is achievable in scientific research, noted Parse co-founder and CEO Alex Rosenberg, Ph.D. Were proud and excited to help support researchers worldwide to reach the ambitious goal of the HCA.

About the Human Cell AtlasThe Human Cell Atlas (HCA) is an international collaborative consortium which is creating comprehensive reference maps of all human cellsthe fundamental units of lifeas a basis for understanding human health and for diagnosing, monitoring, and treating disease. The HCA is likely to impact every aspect of biology and medicine, propelling translational discoveries and applications and ultimately leading to a new era of precision medicine.

The HCA was co-founded in 2016 by Dr. Sarah Teichmann at the Wellcome Sanger Institute (UK) and Dr. Aviv Regev, then at the Broad Institute of MIT and Harvard (USA). A truly global initiative, there are now more than 2,900 HCA members, from 94 countries around the world. https://www.humancellatlas.org

About Parse BiosciencesParse Biosciences mission is to accelerate progress in human health and scientific research. At the core of our company is our pioneering approach for single cell sequencing. Single cell sequencing has already enabled groundbreaking discoveries which have led to new understandings of cancer treatment, tissue repair, stem cell therapy, kidney and liver disease, brain development, and the immune system. At Parse Biosciences, we are providing researchers with the ability to perform single cell sequencing with unprecedented scale and ease. To learn more, please visit https://www.parsebiosciences.com/.

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Parse Biosciences and Human Cell Atlas Partner to Help Map ... - PR Web