Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells

Tristan, Carlos A.; Ormanoglu, Pinar; Slamecka, Jaroslav; Malley, Claire; Chu, Pei-Hsuan; Jovanovic, Vukasin M.; Gedik, Yeliz; Bonney, Charles; Barnaeva, Elena; Braisted, John C.; Mallanna, Sunil K.; Dorjsuren, Dorjbal; Iannotti, Michael J.; Voss, Ty C.; Michael, Sam; Simeonov, Anton; Singeç, Ilyas

doi:10.1101/2020.08.03.235242

Cited by 10 publications

(8 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The Wes and Jes automated Western blotting systems (ProteinSimple) were used for quantitative analysis of protein expression as described previously 20,48 . All western blot data are displayed by lanes in virtual blot-like images.…”

Section: Methodsmentioning

confidence: 99%

Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

Singeç

Jovanovic

Malley

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

Derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome, impeding their use in biomedical research. Here, we developed a highly efficient chemically defined astrocyte differentiation strategy that overcomes current limitations. This approach largely bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro experiments. hPSCs were first differentiated into radial glial cells (RGCs) exhibiting in vivo-like radial glia signatures. Activation of NOTCH and JAK/STAT pathways in bona fide RGCs resulted in direct astrogliogenesis confirmed by expression of various glial markers (NFIA, NFIB, SOX9, CD44, S100B, GFAP). Transcriptomic and genome-wide epigenetic analyses confirmed RGC-to-astrocyte differentiation and absence of neurogenesis. The morphological and functional identity of hPSC-derived astrocytes was confirmed by using an array of methods (e.g. electron microscopy, calcium imaging, co-culture with neurons, grafting into mouse brains). Lastly, the scalable protocol was adapted to a robotic platform and used to model Alexander disease. In conclusion, our findings uncover remarkable plasticity in neural lineage progression that can be exploited to manufacture large numbers of human hPSC-derived astrocytes for drug development and regenerative medicine.

show abstract

Section: Methodsmentioning

confidence: 99%

Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

Singeç

Jovanovic

Malley

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Human cell‐based models based on iPSCs, 3D multicellular aggregate or printed models, and microphysiological systems have all shown promise but our understanding of their predictivity of human responses remains limited. 27 Challenges in cell production and differentiation are considerable, 28 and capacity to model complex in vivo interactions, or long‐term disease states or pharmacological responses, remain areas of needed development.…”

Section: A Research Agenda For the Next Decade Of Translational Sciencementioning

confidence: 99%

Opportunities and challenges in translational science

Austin

2021

Clinical Translational Sci

View full text Add to dashboard Cite

American population remains frustratingly poor overall (4). This failure of translation is not for lack of effort; over $50B are spent on translational efforts in the public and private sectors every year. Opportunities for improvement abound: the cost of developing a new drug is now over $2B, continues to increase faster than inflation, and has increased relentlessly since 1950 despite enormous advances in science (5); the clinical trials process is widely acknowledged to be inefficient (6,7); after a drug or intervention is shown to be useful, its dissemination to all patients who could benefit is slow and variable (8,9), and patient adherence to those interventions remains suboptimal and limits the health benefits of the interventions developed (10). In all, the time required for the end-to-end translational process, from an idea in the lab to a drug or other intervention based on that idea reaching all patients who could benefit, is currently over 20 years, and its success rate is below 1%. Inspiring individual examples of remarkably effective therapies -including enzyme replacement therapies for particular rare diseases, small molecule correctors for cystic fibrosis, PCSK9 inhibitors for elevated cholesterol, and gene therapies for forms of inherited blindness and spinal muscular atrophy -demonstrate what a future therapeutic world might hold. But those successes remain the exceptions, with development times still measured in decades and resulting products having extremely high costs that are arguably unsustainable, and certainly undesirable. And while there have recently been some encouraging signs of improved overall timelines and success rates, productivity (success per unit of effort) and timelines have worsened over the last 40 years, in stark contrast to the remarkable increase in productivity in fundamental research during this time.These divergence have been recognized for some time (11,12), and a variety of efforts have been undertaken to address it. In the private sector, multiple mergers and acquisitions were undertaken in attempt to achieve economies of scale, and the multi-company collaborative TransCelerate Biopharma was formed in 2012 to create platforms for sharing among companies. Public-private partnerships, including the Innovative Medicines Initiative (IMI) in Europe, and the Accelerating Medicines Partnerships (AMP) in the U.S., have produced substantial datasets that are benefitting the entire research ecosystem. In the academic sector, the NIH Roadmap aimed to "redefine the ways that medical research is conducted and, ultimately, how research leads to improvements in health" (13). Among the Roadmap programs were several aimed at the translational divide. The Molecular Libraries Initiative (14) created a robust small molecule assay development, screening, and chemistry probe development capacity in the public sector for the first time, and was remarkably productive during its 10-year lifetime (15). The Clinical and Translational Science Awards (CTSA) program ( 16) has been similarly tra...

show abstract

“…To address this challenge, the establishment of automated and quantitative methods, supported by machine learning and advanced robotics, could assist cell therapy developers tremendously. Some of these advances recently have been reported for the derivation and characterization of iPSCs; however, no major progress has been made in the field of organoids (44)(45)(46). Further advances can only come from a highly collaborative team of biologists, bioengineers, data scientists, and/or experts in process automation and control, again highlighting the importance of breaking down silos and enabling cross-disciplinary collaborations.…”

Section: Quality Control Expertsmentioning

confidence: 99%

Intestinal organoids: roadmap to the clinic

Kasendra

Troutt

Broda

et al. 2021

American Journal of Physiology-Gastrointestinal and Liver Physi

View full text Add to dashboard Cite

Recent advances in intestinal organoid research, along with encouraging preclinical proof-of-concept studies, have revealed significant therapeutic potential for induced pluripotent stem cell (iPSC) derived organoids in the healing and replacement of severely injured or diseased bowel [1-3]. To fully realize the tremendous promise of stem cell organoid-based therapies, careful planning aligned with significant resources and efforts must be devoted demonstrating their safety and efficacy to meet critical regulatory requirements. Early recognition of the inherent preclinical and clinical obstacles that occur with the novel use of pluripotent stem cell derived products will accelerate their bench to bedside translation [4-6]. To overcome many of these hurdles, a close and effective collaboration is needed between experts from various disciplines, including basic and clinical research, product development & manufacturing, quality assurance & control, and regulatory affairs. Therefore, the purpose of this article is to outline the critical areas and challenges that must be addressed when transitioning laboratory-based discovery, through an Investigational New Drug (IND) application to first-in-human clinical trial, and to encourage investigators to consider the required regulatory steps from the earliest stage of the translational process. The ultimate goal is to provide readers with a draft roadmap which they could use while navigating this exciting regenerative medicine therapeutic space.

show abstract

Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells

Cited by 10 publications

References 62 publications

Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

Opportunities and challenges in translational science

Intestinal organoids: roadmap to the clinic

Contact Info

Product

Resources

About