5 Days of Stem Cells Virtual Event—2022 Speakers

Dr. Wu received a medical degree from the Beijing Medical University (Peking University Health Science Center) in 1984, and Ph.D. from the University of Texas Medical Branch (UTMB) at Galveston in 1991. After receiving a post-Doc training in the University of Florida, she served as an Instructor at the Harvard Medical School and Beth Israel Deaconess Medical Center. In 1999, Dr. Wu was recruited as a tenure-track Assistant Professor in the Department of Anatomy and Neurosciences at UTMB. She is currently a tenured Full Professor and Vice Chair for Research in the Department of Neuroscience, Cell Biology and Anatomy. Dr. Wu is also an Investigator of the George P. and Cynthia Woods Mitchell Center for Neurodegenerative Diseases, the Center for Addiction Research, the Institute for Human Infections and Immunity, the Moody Center for Traumatic Brain & Spinal Cord Injury Research, and the TIRR Mission Connect. Dr. Wu has been the inaugural holder of the John S. Dunn Distinguished Chair in Neurological Recovery since 2005. She serves as Associated Editor, Section Editor or Editorial board member on several scientific journals, as well as council members on several scientific societies or associations. Dr. Wu’s current study focuses on exploring the biology and therapeutic potentials of human and rodent neural stem cells. Her translational research interests include neurotrauma, addiction and neural infection.

Culture of human fetal brain-derived neural stem cells (hNSCs) provides an excellent tool to investigate mechanisms by which viral infection affects human brain development and to test potential drugs that may mitigate viral toxicity. Here I will briefly introduce two methods to generate neurons and glia from hNSCs. Next, I will present our findings on using hNSCs to decipher mechanisms underlying Zika virus (ZIKV)-associated abnormal neural development. ZIKV infection causes microcephaly in some infants born to infected pregnant mothers. Our data suggest that ZIKV-induced abnormal neurogenesis of hNSCs is dependent upon human individual responses, cell stages, and viral strains. Furthermore, ZIKV infection over activates innate immune responses of hNSCs. Our study suggests that orchestrating the host innate immune responses after ZIKV infection could be a promising therapeutic approach to attenuate ZIKV-associated neuropathology.

Dr. Loh Yuin-Han Jonathan is currently a Research Director at the A*STAR Institute of Molecular and Cell Biology (IMCB), and a Program Director for A*STAR Cell Therapy and Manufacturing research. His lab in IMCB focuses on 1) identifying the global regulatory and metabolic switches in stem cell maintenance and during cell-fate transition, and 2) the development of novel tools, technologies, and innovations for therapeutic applications. In so doing, the team has developed expertise in the domain of connecting epigenetic regulations to cell fate determinations (Cell 2015, Nature Communications 2019). Over the years, their research has elucidated the molecular mechanisms underpinning Hematopoietic stem cells (HSCs) renewal and their exit to a differentiated state (Nature Communications 2018, Nature Communications 2016, Nature Cell Biology 2022). Furthermore, the team has deconstructed the pluripotency and cell-fate reprogramming process using gene engineering, genome topology, and lab-developed single-cell analytical tools (Nature Methods 2016, Science Advances 2020, Genome Research 2020, Nucleic Acid Research 2022). Till date, Jonathan's publications have been cited 20,411 times (Google Scholar) by peers worldwide. Jonathan's research work has earned him several prestigious national and international accolades including the Singapore National Academy of Science Young Scientist Award, Singapore Youth Award, A*STAR Investigatorship Award, World Technology Network Fellowship, MIT TR35 Asia Pacific Award, Stem Cell Society Singapore Outstanding Investigator Award and the National Research Foundation Investigatorship Award. He serves on the Education Committee of the International Society Singapore (SCSS) and the Executive Council for the Singapore Association for the Advancement of Science (SAAS).

Stem cells and lineage tissues are defined by their unique gene expression profiles, which in turn is regulated by dynamic epigenetic processes including DNA methylation chromatin medications and genome topology. In this talk we will share our genomic survey of SETDB1 binding in mouse embryonic stem cells which led to the unexpected discovery of regions bereft of common repressive histone marks (H3K9me3, H3K27me3). Further profiling of these non-H3K9me3 regions led to the discovery of the cluster of non-repeat loci that were co-bound by SETDB1 and Cohesin. These regions, which we named DiSCs (Domains involving SETDB1 and Cohesin) were seen to be proximal to the gene promoters involved in embryonic stem cell pluripotency and lineage development. Importantly, it was found that SETDB1-Cohesin co-regulate target gene expression and genome topology at these DiSCs. Depletion of SETDB1 led to localized dysregulation of Cohesin binding thereby locally disrupting topological structures. Dysregulated gene expression trends revealed the importance of this cluster in ES cell maintenance as well as at gene 'islands' that drive differentiation to other lineages. We will further discuss our recent finding on TE (Transposable element) associated enhancers in regulating stem cells. We identified global TE signature profiles across the different potency states (2i, ground and expanded potential) and further characterized the role of a nuclear receptor transcription factors which regulates the action of the TE-enhancer switches.

Dong received his Ph.D. in Cell Biology from Peking University, Beijing, China. He identified RNF135/RIPLET/REUL as an essential E3 ligase of RIG-I in antiviral immunity. In 2012, Dong joined the laboratory of Dr. Yu Chen as a postdoctoral fellow at Memorial Sloan Kettering Cancer Center, New York. There, he established the patients derived prostate cancer organoids culture system. These prostate cancer organoid models will help us address the basic scientific questions and identify novel therapeutic strategies of prostate cancer. In 2016, Dong joined the faculty at Shanghai Institute of Biochemistry and Cell Biology as a principal investigator. The main goal of Dong’s research group is understanding the mechanisms of cancer cell lineage plasticity in response to genetic/epigenetic alterations and targeted therapies. Dong is the recipient of a Distinguished Young Scholars Fund of NSFC (2021), a Program of Shanghai Subject Chief Scientist (2021), and a Thousand Young Talents Awards (2016).

Cancer initiation, progression and targeted therapeutic resistance are often associated with cell lineage plasticity which are recognized as novel mechanisms of therapeutic resistance. Exemplified adeno-to-neuroendocrine lineage transitions have been identified in PCa and lung adenocarcinoma following the application of molecularly targeted therapies involving AR inhibition and tyrosine kinase inhibition, respectively. However, the dynamics of both de novo and targeted therapy-associated lineage plasticity are largely unknown. Therefore, systematically characterizing the genetic, epigenetic, and microenvironmental factors responsible for this adeno-to-neuroendocrine lineage transition may reveal the underlying molecular details and stimulate the development of novel targeted therapies to prevent or reverse resistance. Identifying the direct molecular drivers and developing pharmacological strategies using clinical-grade inhibitors to overcome current lineage transition-induced therapeutic resistance are imperative. While cancer is commonly perceived as a disease of dedifferentiation, the hallmark of early stage prostate cancer is paradoxically the loss of more plastic basal cells and the abnormal proliferation of more differentiated secretory luminal cells. However, the mechanism of prostate cancer pro-luminal differentiation is largely unknown. We identified the essential role of ERG in regulating prostate cell lineage toward a proluminal program by orchestrating chromatin interactions. Using single-cell multiomics analyses, we investigated the dynamics of cellular heterogeneity, transcriptomics regulation and microenvironmental factors in 107,201 cells from mouse prostate cancer samples with complete time series of tumor evolution seen in patients. These results have the potential to reveal the drives of adeno-to-neuroendocrine lineage plasticity in prostate cancer, and provide potential pharmacological strategies for castration-resistant NEPC.

Dr. Jun Wu is an assistant professor in the department of molecular biology at UT Southwestern Medical Center. Dr. Wu’s work has contributed to the development of novel culture systems and methods that enable the generation of new stem cells for basic and translational studies. Dr. Wu has expanded the spectrum of pluripotent states by capturing mouse pluripotent stem cells (PSCs) with distinct molecular and phenotypic features from different developmental stages. And some of these culture conditions developed in mice enabled the generation of PSCs from many other mammalian species, including humans, non-human primates, and ungulates. In addition, Dr. Wu has developed an efficient and versatile blastocyst complementation system for in vivo generation of functional tissues and organs from cultured PSCs, and several stem cell derived blastocyst models (blastoids). Dr. Wu has received several awards including UT southwestern endowed scholar, CPRIT scholar and NYSCF-Robertson Stem Cell Investigator award.

Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Provided with the right combination of factors promoting self-renew while shielding them from differentiation, transitory pluripotent epiblast cells in vivo can be propagated indefinitely in culture as pluripotent stem cells (PSCs). Recent advances in derivation of PSCs with distinct molecular and phenotypic features open new avenues for regenerative medicine and developmental biology. During my presentation, I will elaborate on the use of PSCs for two research directions: interspecies blastocyst complementation and stem cell embryo models.

Interspecies blastocyst complementation for organ generation:

Shortage of organs for transplantation is one of the largest unmet medical needs. PSCs offer a potential unlimited source of donor organs. Despite years of research, however, it remains infeasible to generate organs from PSCs in vitro as the interactions among cells and tissues during development and organogenesis are too complex to reproduce. An in vivo approach known as interspecies blastocyst complementation, which enables the formation of an organ from one species inside another species, holds potential to overcome this hurdle. One of the keys to success for interspecies blastocyst complementation is the ability of PSCs from the donor species to contribute to chimera formation in the host species. Rat and mouse PSCs can robustly contribute to chimera formation in mouse and rat, respectively, which enabled the generation of rat pancreas, thymus, embryonic heart and endothelial tissues in mice, and mouse pancreas and kidney in rats. To date, however, robust chimerism between distantly related species has not been achieved even at embryonic stages, suggesting a xenogeneic barrier exists between evolutionary distant species during early development. Cell competition, the process of eliminating viable but “less fit” neighbor cells, has been proposed as a surveillance mechanism to ensure normal development and maintain tissue homeostasis. During interspecies chimera formation, cells from the donor species may be treated as unfit or aberrant cells targeted for elimination. We developed an interspecies PSC co-culture strategy and uncovered a previously unrecognized mode of cell competition between evolutionarily distant species. We discovered that cell competition constitutes a major component of the xenogeneic barrier and overcome interspecies pluripotent cell competition improves chimerism between evolutionary distant species.

Human blastoids:

Our cellular, molecular and genetic understanding of human pre-/peri-implantation development is limited. This gap in knowledge is a key obstacle to understanding the basis of developmental defects. However, studying early human development is challenging due to the restricted access to human embryos available for research. Rodent models play an important role in understanding early human development, where a powerful array of modern genetic tools has been successfully applied. However, rodent models are not ideal because many aspects of human development are molecularly and temporally distinct from the rodents. To relieve the dependency of human embryos for studying human embryogenesis, last year we first reported a method for the generation of 3D human blastocyst-like structures (termed human blastoids) from naive human pluripotent stem cells (hPSCs), which recapitulate all three embryonic tissue types (Yu et al., Nature, 2021). We have further improved the method, which now supports highly efficient (~80%) and large-scale production (>20,000 per plate) of human blastoids. Human blastoids represent a well-controlled cellular substrate to test biological hypotheses and will facilitate an improved fundamental understanding of the key signaling events and cellular interactions of pre-/peri-implantation human development, which will help us understand the molecular and cellular contributors of developmental failures in humans.

Coming soon

Solid tumors have remained particularly refractive to immunotherapies due to a severely immunosuppressive microenvironment, heterogeneity and functional and metabolic reprogramming, which in turn renders immune effector cells, such as natural killer (NK) cells, hypofunctional. Leveraging the ability of induced pluripotent stem cells to generate phenotypically mature and functionally competent NK cells can lead to potent off-the-shelf immunotherapies. When combined with genetic engineering approaches, such gene-modified, stem cell-derived immune effectors can effectively and potently target severely immunosuppressive mechanisms of tumor resistance and NK cell immune activity, including metabolic inhibition via purinergic signaling, antigen escape, and checkpoint-induced immunosuppression, such as that driven by TIGIT. This talk will explore how the generation of genetically-engineered, stem cell-derived NK cells can result in durable anti-tumor responses when targeted against multiple mechanisms of immunosuppression in solid tumors. The talk will also discuss these cells as platforms for future off-the-shelf immunotherapies.

Dr. B.C. Das has made outstanding contributions in the field of Cancer Research, Tumor Virology, Human Genetics and Mutation Research. During the last 45 years of his distinguished research career he has published more than 250 research papers in reputed international journals and distinguished himself as a renowned Molecular Oncologist. Dr. Das has earned his M.Sc. and Ph.D. degree from Banaras Hindu University, Varanasi. Later he worked several years with the Nobel Laureate, Prof. Harald zurHausen (Nobel Prize, 2008) at German Cancer Research Centre (DFKZ), Heidelberg, (Germany) and in India he pioneered the work on Human Papillomavirus (HPV). He established National and WHO Regional HPV Referral Centre for South-East Asia at ICMR-NICPR. His present areas of research are molecular epidemiology, transcriptional regulation, miRNA regulation, cancer stem cell, targeted drug delivery, drug development and gene editing.

BACKGROUND

The principal etiologic agents that cause cancer of the uterine cervix in women is persistent infection of high-risk Human Pappillomaviruses (HR-HPVs), but HPV alone is not sufficient to cause cancer. Though there are three protein-based prophylactic HPV vaccines and now an indigenous Indian HPV vaccine, Cervavac, but no therapeutic vaccine is available. With an extensive molecular epidemiologic study and national mapping of HPV and their genome sequencing, we have developed India specific HPV-16 DNA variant-vaccine construct as an indigenous therapeutic-cum-preventive, a chimeric DNA vaccine for an effective prevention and treatment of cervical cancer. Though more than 110 countries world over introduced HPV vaccine successfully in their national program but in India in spite of high annual incidence of HPV and cervical cancers and death due to cervical cancer, there is no initiative till date to incorporate HPV vaccine in national immunization program in India.

HR-HPV E6/E7 is responsible for tumorigenic transformation and their expression depends on the availability of host cell transcription factor (AP-1), which binds to the upstream regulatory region (URR) of high-risk oncogenic HPVs and act as a signaling epicenter for cervical cancer. It is also known that a small sub-population (1 to 2%) of cancer stem cells (CSCs) are responsible for tumor initiation, progression, metastasis, treatment resistance and disease relapse even after successful treatment. We have, therefore, attempted to develop a novel triple conjugate drug for targeted delivery to cancer and cancer stem cells without affecting the normal cells which otherwise cause serious adverse/toxic side effects.

MATERIALS AND METHODS

Standard molecular biology methods and stem cell culture, sphere formation, immunoblotting, Realtime PCR, mouse tumorigenicity testing, immunohistochemistry band-shift assay and synthesis of triple conjugate drug, clinical validation including stem cell targeting experiments and bioavailability assays were carried out.

RESULTS

HPV E6 is found to be differentially upregulated in CSCs and is responsible for maintenance of stemness through upregulation of Hes1, a downstream gene of stemness marker NOTCH1. AP-1 overexpression contributes to chemoradioresistance of CSCs which can be sensitized if fra-1 (Fos related antigen 1), one of the family proteins of AP-1 is upregulated. A triple conjugate drug comprising curcumin-folic acid -cancer drug (Doxorubicin) has been synthesized as a non-toxic, small molecular weight novel drug which will specifically be taken out by the cancer and cancer stem cells via receptor-mediated internalization due to over-expression of high-affinity folate receptor in these cells thereby improving the bioavailability and targeted delivery of the drug. While folic acid serves as a targeting ligand, curcumin chemoradiosensitizes the CSCs and also reduces the toxicity and adverse effects thus making the cancer treatment most effective and relapse free.

CONCLUSION

The novel Curcumin-Folate and standard cancer drug conjugate potentially ensure sensitization of CSCs making the cancer treatment most effective.

Coming soon

At this virtual conference, I would like to introduce you to the Parkinson's disease cell therapy using pluripotent stem cells conducted by S.Biomedics Co., Ltd. Clinical research on Parkinson's disease cell therapy using pluripotent stem cells is actively underway in the United States, Japan, and Europe. One of the main issues with Parkinson's disease cell therapy is the strategy for how efficiently A9 dopamine neurons can be supplied. The S.Biomedics research team has developed a technology that efficiently differentiates A9 dopamine neuron progenitor cells from human embryonic stem cells and has currently applied for approval from the Korea Ministry of Food and Drug Safety for a 1/2a clinical trial. In this conference, I would like to report on the differentiation strategy and clinical development status of S.Biomedics.

Coming soon

We developed a fully defined, clinically compatible cell culture system that can generate purified, functional, and therapeutically effective endothelial cells (ECs) from human pluripotent stem cells (hPSCs), which include human embryonic stem cells and induced pluripotent stem cells. We further encapsulated hPSC-ECs within the nanomatrix gel and transplanted them into experimental hindlimb ischemia. These encapsulated hPSC-ECs remained engrafted for more than 10 months in ischemic tissues, and when compared to bare hPSC-ECs, they exerted higher and prolonged neovascularization and showed better vascular regenerative capacity.

Direct conversion or reprogramming of human postnatal cells into ECs, bypassing stem or progenitor cell status, is crucial for cell therapy, and pathophysiological investigation but has remained largely unexplored. We thus sought to directly reprogram human postnatal dermal fibroblasts (HDFs) to ECs with vasculogenic and endothelial transcription factors (TFs) and determine their vascularizing and therapeutic potential. We found that ER71/ETV2 alone is able to directly reprogram human postnatal cells to functional, mature ECs, referred to as reprogrammed ECs (rECs). These rECs could be valuable for cell therapy, disease investigation, and exploration of the reprogramming process.

MD, PhD, Researcher at Fiocruz and D'Or Institute for Research and Education.

Medical director at the Center for Biotechnology and Cell Therapy, São Rafael Hospital, in Salvador, Brazil.

The field of cell & gene therapy is evolving rapidly and mesenchymal stromal cells (MSCs) are currently under clinical investigation for several conditions due to their immunomodulatory and trophic actions. Extracellular vesicles (EVs) are fundamental effectors of MSCs, playing a role in intercellular communication and in the regulation of several biological processes, that favor the control of hyperinflammatory states and the promotion of tissue repair. In the past years, with a growing interest in exploring the potential translation of EVs into “cell-free” therapeutics, many challenges to the establishment of standards and cGMP-compliant protocols for the manufacturing of such products have been faced and many advances were made, leading to the regulatory approval of the first clinical trials. This presentation reviews basic concepts of MSC-EV biology, and current standards in terms of characterization and discusses the group experience in validation of cGMP manufacturing of MSC-EVs and nonclinical evaluation of toxicity and efficacy.

I am an entrepreneur with a major in biology, a PhD in human genetics and stem cell biology and an MBA in health innovation management. I have co-founded two biotech companies. I am currently CEO of LizarBio Therapeutics, a cell therapy company developing curative treatments for incurable diseases.

Induced pluripotent stem cell technology (iPSC) has been used to derive several different types of cells, including cardiomyocytes, that can be used for therapeutic applications. Cardiomyocytes are a relevant component of the heart tissue and many patients suffering from heart failure could potentially benefit from heart cell replacement therapy. Here we share our successful results of how we reprogrammed adult cells into iPSCs, differentiated them into bonafide immature human cardiomyocytes and assessed their ability to functionally improve myocardial infarcted rats and resmuscularize pig hearts.

Dr. Li is the professor of Zhongshan Medical College of Sun Yat-Sen University. He was selected into the 2015 Guangdong Special Support Program Hundreds of Thousands of Young Talents Training Program, the 2015 Sun Yat-Sen University Outstanding Young Teacher Training Program, the 2014 Guangzhou "Pearl River Science and Technology Rising Star" Special Program, and the 2012 Guangdong Provincial Higher Education "Thousand Hundred Ten Projects" "The seventh batch of school-level training objects, as an instructor, participated in the 2012 "2nd National Undergraduate Basic Medical Innovation Forum and Experiment Design Competition" and won the second prize, and won the "2010 Outstanding Postdoctoral Fellow of Sun Yat-sen University".

Robust protocol for generation of NC-PC from Hpsc

• NC-PC possessed typical phenotype of pericytes
• In vivo enrichment of NC-PC could improve neurological function and protect neurons from death by preserving BBB integrity and expressing MDK in AID model
• NC-PC may represent an ideal cell source for the treatment of BBB dysfunction-related disorders

James graduated from UC San Diego in 2017 with a bachelor’s in Physiology & Neuroscience and spent the next two and a half years working in the Stem Cell Core of the Salk Institute for Biological Studies in La Jolla, California. His research there surrounded testing and validating new media systems in iPSC culture, generating new iPSC lines from dermal punch biopsies and training new scientists on hPSC culture techniques. In late 2019 he made the transition to industry and joined Thermo Fisher Scientific’s Custom Services Biology team in Carlsbad, California, where he helped lead the generation of a large iPSC cohort through reprogramming of PBMC patient samples. Since then, his work has focused on iPSC genome editing, lentivirus-based cell line generation, and transcription factor mediated stem cell differentiation.

Human induced pluripotent stem cells (hiPSCs) have been globally recognized as a multipurpose research tool for modeling human disease and biology, screening and developing therapeutic drugs, and implementing cell therapies. The ability to differentiate human iPSCs into any cell type supports an unlimited source for the generation of cell replacement therapies. The emergence of genome editing tools, including the CRISPR/Cas9 system or TALENs enable genetic modification of the genome of these cells, allowing knockouts or transgene insertions to alter biological functions of the desired differentiated cell type for allogeneic cell therapy applications. 

While genome engineering of hiPSC has become relatively straightforward, reagents and workflows generation of genome edited hiPSC in a cGMP environment are currently scarce. Using the generation of immune evasive (allogeneic) hiPSC through genome engineering as an example, we showcase a workflow that implements next generation reagents and instruments that enable cGMP manufacturing of engineered hiPSC. We demonstrate that through the use of novel cGMP grade reagents, genome editing in hiPSC is highly efficient, and survival and genomic stability of edited cells through the genome editing workflow is maintained throughout the workflow. We furthermore demonstrate that available closed systems perfectly integrate into this workflow.

In summary, we detail advances with tools, reagents and protocols that facilitate the genome editing workflow in hiPSC in a cGMP environment and demonstrate that the use of such tools can be readily implemented in generation of hiPSC for clinical application, with applications in manufacturing of allogeneic cell therapy products.

Connie Lebakken is a Co-Founder and the Chief Operating Officer of Stem Pharm. She has deep experience in the development of high-throughput biochemical and cell-based drug discovery assays, and tools and protocols to improve stem cell workflows. The team at Stem Pharm is focused on utilizing synthetic hydrogels to improve neural cell culture and neural organoid formation and applying transcriptional and proteomic readouts to study neuroinflammation and neural toxicities. Dr. Lebakken received a PhD in Cellular and Molecular Biology from the University of Wisconsin and postdoctoral training in Physiology and Biophysics at the University of Iowa.

Advances in three-dimensional culture systems have enabled the development of models that better capture the 3D nature of native tissue. Animal-derived extracellular-matrix products are commonly used for encapsulation or as scaffolds for these 3D models; however, these materials are generally undefined, variable, and have safety concerns due to their animal origin. Defined synthetic materials allow for more precise design and optimization of substrates for specific cellular applications that are free from animal derived components. Through control of properties such as substrate mechanical stiffness and adhesion ligand presentation, materials can be designed that enable more physiologically relevant extracellular environments and better direct cellular behaviors within 3D models. At Stem Pharm, we are applying these principles to design PEG-based hydrogels for the development of in vitro neural models. We have developed methods to generate thin hydrogel coatings that can be applied to standard tissue-culture surfaces to improve assay metrics for neural applications. These surfaces can be pre-coated and delivered sterile and ready to use. We have utilized our PEG-based hydrogels to generate novel neural organoids that incorporate microglia and can be used to study neuroinflammation and neurotoxicity. We will present case studies highlighting these applications.

Dr. Zhiying Guo is the senior R&D scientist in Beijing CELLada Biotechnology Co., LTD., now. She researched the stem cell biology, tumor biology, and organoids for a long time. And has rich experience in the field of the organoid model construction, including the adult stem cell derived organoids and the PSC-derived organoids.

Stem cells have the potential of self-renewal and proliferation. They can develop into many different cell types. There are several main categories: the pluripotent stem cells and somatic stem cells. Pluripotent stem cells have the ability to differentiate into all of the cells of the adult body. Somatic stem cells can differentiate to the specialized cell types of the organ. In recent years, various organoids have been generated from both pluripotent stem cells (PSCs) and adult stem cells (ASCs) by mimicking the intestine, liver, lung, brain and pancreas. Organoids have organ-specific structure and function. Our group constructed the organoids of intestinal, stomach, lung, mammary gland, esophagus, and glioma from adult primary tissues. Patient-derived organoids can be used for drug sensitivity testing and clinical precision medicine. From iPSC and embryonic stem cells, the heart, blood vessels, brain, liver, kidney, lung and small intestine organoids are created, which used to research the development and the mechanism of diseases.

Timothy is the Principal Investigator and Associate Professor in the departments of Cell, Development and Regenerative Biology and Ophthalmology, member of the Black Family Stem Cell Institute at the Icahn School of Medicine at Mount Sinai. He was a Hong Leong visiting professor at the National University of Singapore from 2017 to 2019 and a current visiting professor at the University of Chieti. He received his PhD from New York University Medical Center under the mentorship of Dr. Eric Lang, directed by Rodofo Llinàs. He then became a post-doctoral fellow at the Neural Stem Cell Institute under the leadership of Dr. Sally Temple. Dr. Blenkinsop leads a team focused on understanding the plasticity of the retina in the effort to develop therapies for retina-based eye diseases. He is a member of the Association for Research in Vision and Ophthalmology and International Society for Stem Cell Research. Dr. Blenkinsop has authored publications in journals including Cell Stem Cell, Nature Communications, Journal of Neuroscience and Stem Cell Reports, which have been highlighted in Nature in Cell. His lab has been written about in the press and has been interviewed by new outlets including the Ophthalmology Times. He also consults with companies in the field of neural stem cells, immunosuppression and retinal regeneration. Dr. Blenkinsop has won awards for mentorship as wells as imaging competitions and honorariums. His laboratory focuses on the development of strategies for repairing or regenerating the retina by both cell replacement and endogenous regeneration. His work has led to the first in man transplantation of adult human cadaver derived RPE into patients.

The therapeutic potential of pluripotent stem cells is great as they promise to usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop this technology as it lends itself to in vivo monitoring and sophisticated non- invasive imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with several eye diseases. However, after transplantation, the cells may transform and cause complications. This transformation may be partially due to incomplete maturation. With the goal of learning how to mature RPE, we compared induced pluripotent stem cell- derived RPE (iPSC-RPE) cells with adult human primary RPE (ahRPE) cells and the immortalized human ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and ahRPE cells for one month, and evaluated morphology, RPE marker staining, and transepithelial electrical resistance (TEER) as quality control indicators. We then isolated RNA for bulk RNA-sequencing and DNA for genotyping. We genotyped ahRPE lines for the top age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) risk allele polymorphisms. Transcriptome data verified that both adult and iPSC-RPE exhibit similar RPE gene expression signatures, significantly higher than ARPE-19. In addition, in iPSC-RPE, genes relating to stem cell maintenance, retina development, and muscle contraction were significantly upregulated compared to ahRPE. We compared ahRPE to iPSC-RPE in a model of epithelial-mesenchymal transition (EMT) and observed an increased sensitivity of iPSC-RPE to producing contractile aggregates in vitro which resembles incident reports upon transplantation. P38 inhibition was capable of inhibiting iPSC-RPE–derived aggregates. In summary, we find that the transcriptomic signature of iPSC-RPE conveys an immature RPE state which may be ameliorated by targeting “immature” gene regulatory networks.

Dr. Forbes received his doctorate from The George Washington University where he studied factors influencing oligodendrocyte maturation, myelination, and functional recovery after perinatal brain injury. Thomas continued his postdoctoral work at Children's National Hospital in Washington, DC, where he focused on social, physical, and cognitive enhancement of surroundings to ameliorate neurodevelopmental insult. Here, he uncovered molecular mechanisms involved in enrichment-induced recovery and demonstrated that myelin plasticity induced by modulation of the neonatal environment can be targeted as a therapeutic strategy for preterm birth. Thomas currently resides in Carlsbad, CA and has been with Thermo Fisher Scientific for two years.

Full differentiation of human pluripotent stem cells into mature neural organoids is a significant advancement in stem cell research. These organoids serve as neuron banks that can be dissociated and used for disease modeling, drug discovery, and cell therapy. Neural organoids developed for specific applications should have regional specificity and express developmental markers characteristic of human brain cells. Here, we discuss how human PSCs cultured into aggregates in suspension can be successfully differentiated into functional forebrain-specific organoids. This protocol is compatible with defined and enriched media systems and allows for robust expression of mature forebrain markers in as few as 28 days. After completion of neural induction, cell aggregates can be dissociated and expanded in two dimensions to increase the neuron yield prior to maturation. Additionally, dorsal- and ventral-specific forebrain organoids can be obtained, and cultures in both 3D and 2D formats demonstrate robust electrical activity. Neural organoids provide an opportunity to study the complex biology of the brain in a physiologically relevant manner, and therefore constitute a powerful tool for human-specific in-vitro modeling of neurological development and disorders.

Rupa is the Sr. Director of Technical Affairs for Advanced Therapies at Thermo Fisher Scientific. The Office of Technical Affairs comprises scientific experts that serve as a strategic, innovational and educational leaders in the area of cell-based therapies, plasmids and mRNA therapeutics. In her prior role as the Director of Enterprise Science and Innovation Partnerships, she developed and managed strategic partnerships with global BioPharma, Biotech and Healthcare customers in the area of Cell and Gene Therapy. Prior to this, she was the Head of Technical Operations (Patheon/Thermo Fisher Scientific) where she worked closely with customers to conduct technology transfer and process optimization activities related to GMP manufacturing of cell-based therapies. She has over 15 years of expertise in GMP manufacturing and has successfully led GMP operations, Process Development and MSAT activities, infrastructure buildout, customer relations and business development. In her past roles, she has been the Director of Cell Manufacturing for Program for Advanced Cell Therapy- UW Hospitals and Clinics. At Ligand Pharmaceuticals, she was part of the team that designed novel hematopoietic screening assays for erythropoietin and thrombopoietin in partnership with GlaxoSmithKline, a part of the program that led to development of Promacta®.

Can Induced Pluripotent Stem Cells Accelerate Off-the-shelf Allogeneic Cell Therapies?

Cell-based therapies have shown unprecedented promise for life-changing treatment options. While autologous therapies have rapidly advanced with multiple commercial products on the market, they are often accompanied by complex supply chain and manufacturing logistics. Additionally, autologous therapies can be expensive, difficult to scale and not easily accessible. As a result, many manufacturers have begun to focus on allogeneic therapies using donor-derived starting materials such as bone marrow, cord blood and induced pluripotent stem cells (iPSCs). Allogeneic therapies, using cells from healthy donors, have the potential to overcome many of the limitations posed by autologous therapies and generate “off the shelf” products that are far-reaching and affordable.

iPSCs have emerged as a favorable cell type and can be used as starting material both for autologous and allogeneic therapies. Significantly more efforts are being dedicated to iPSC- derived allogeneic treatments such as CAR-T and CAR-NK, as many doses can be manufactured simultaneously from a single batch of iPSCs. The manufacturing process can be scaled up to achieve economies of scale and reduce high costs. Through simplified logistics and minimizing the time to reach patient, iPSCs can accelerate “off-the-shelf” allogeneic therapies for patients with unmet medical needs.

Michael Akenhead is an R&D Scientist at Thermo Fisher Scientific. His primary research involves pluripotent stem cells (PSCs), particularly related to efficient PSC expansion and subsequent use of PSCs in downstream applications. His current research has focused on expanding PSCs as spheroids in 3D suspension cultures. Michael Akenhead holds a PhD in biomedical engineering from the University of Kentucky, where he performed research on neutrophil mechanobiology. After completion of his dissertation, he came to Thermo Fisher Scientific and began his research involving PSCs.

The ability to scale up to large quantities of cells is necessary for many therapeutic and screening applications. We have utilized StemScale PSC Suspension Medium to support the growth of pluripotent stem cells as spheroids in liter-scale bioreactors. StemScale can rapidly generate billions of cells within a short amount of time. Although it is often cumbersome to process the cell yields from large-scale bioreactors, we developed a protocol to pair the Rotea Counterflow Centrifugation System with StemScale spheroids. The Rotea can rapidly dissociate these spheroids and harvest the resulting single cell output, all while maintaining cells in a closed system environment. Furthermore, the Rotea can be adapted for other applications involving StemScale, such as assisting in medium exchanges which support the downstream differentiation of spheroids.

Helen Miranda graduated with a BS in Biomedicine and a MS in Immunopathology from the State University of Londrina In Brazil. She then went to earn her PhD in Cell and Molecular Biology in the University of Sao Paulo, also in Brazil. As a graduate student, Helen was one of only 10 Brazilians selected by the renowned stem cell biology experts in the UK for the “Embryonic Stem (ES) Cells as a Model System for Embryonic Development” course. During this experience, she became fascinated by the use of induced pluripotent stem cells (iPSCs) for modeling human diseases. For that reason, Helen chose to come to the USA as a joint postdoctoral fellow in the La Spada lab and in the Muotri lab at the University of California San Diego (UCSD). During her postdoctoral training, Helen developed stem cell models for two different motor neuron disorders, spinal bulbar muscular atrophy and amyotrophic lateral sclerosis. She joined the Department of Genetics and Genomes Sciences at Case Western Reserve University as an Assistant Professor in May 2018 to continue her studies on the pathophysiology of motor neuron diseases.

Neuromuscular junctions (NMJs) are specific synapses that connect motor neurons to skeletal muscle and therefore are primarily responsible for voluntary movement. The degeneration of this system can lead to symptoms that are observed in motor neuron and neuromuscular diseases. However, the knowledge of human NMJs is scarce due to obvious ethical concerns and the relative inaccessibility of samples. In this work, we use human-induced pluripotent stem cells (iPSCs) to generate motor neurons and skeletal muscles and co-culture these two cell types to form an in vitro humanized NMJ system. IPSC-derived NMJs were characterized morphologically by a-bungarotoxin staining. We also observed that when iPSC-derived skeletal muscles and motor neurons were co-cultured in the presence of agrin, there was a significant increase in the expression of acetylcholine receptor (AChR). Furthermore, we performed functional analysis of the iPSC-derived motor neuron and skeletal muscles separately by a multielectrode array (MEA) system using the Maestro instrument (Axion Biosystems). Both isolated cultures display spontaneous spike activity that increases over time, indicative of maturation of the cultures. The quantification of those enabled the optimization of the iPSC-derived NMJs functional analyses using the MEA system. In the iPSC-motor neuron and skeletal muscle co-culture, we observed increased spike frequency when compared to skeletal muscle alone. We have also taken advantage of optogenetics and transduced the iPSC-derived motor neurons with a lentivirus harboring the humanized channelrhodopsin (ChR2) under the control of the synapsin promoter. Transduced motor neurons co-cultured with skeletal muscle displayed an increase in Bursts when subjected to light stimulation using the LUMUS apparatus (Axion Biosystems). To our knowledge, we are the first laboratory to optimize the MEA system for quantitation of iPSC-derived skeletal muscle activity and to measure functional human NMJs. We are currently using this system to investigate NMJ phenotypes associated with motor neuron disease pathogenesis.

Evangelos Kiskinis PhD is an Assistant Professor of Neurology and Neuroscience at Northwestern University Feinberg School of Medicine and a New York Stem Cell Foundation Robertson Investigator. Dr. Kiskinis earned a PhD from Imperial College London and carried out postdoctoral training at Harvard University where he pioneered some of the first models of amyotrophic lateral sclerosis (ALS) using personalized stem cell-based approaches. His laboratory utilizes patient-specific induced pluripotent stem cells (iPSCs) and reprogramming approaches to develop models of neurological diseases such ALS and pediatric forms of severe neurodevelopmental epilepsy syndromes. Their core interest is to understand how distinct neuronal subtypes develop and how they become dysfunctional and degenerate during injury or disease. The overarching goal of his research is to provide novel insights into neuronal development and identify points of targeted and effective therapeutic intervention for epilepsy and ALS. At Northwestern he also serves as the Scientific Director of the Stem Cell Core Facility.

Human induced pluripotent stem cell (iPSC) technologies offer a unique resource for modeling neurological diseases. However, iPSC models are fraught with technical limitations including abnormal aggregation and inefficient maturation and ageing of differentiated neurons. These issues are in part due to the absence of synergistic cues derived from the architecture, chemical composition and molecular dynamics of the native extracellular matrix (ECM). To solve these problems we purified the ECM from the spinal cord of early postnatal and adult mice and profiled its protein composition by mass spectrometry (MS)-based proteomics. We identified several candidate proteins that were significantly more abundant in aged ECM, including laminin alpha 1 chain. We hypothesized that laminin alpha 1, which engages with neuronal surface receptors through the bioactive pentapeptide sequence IKVAV, might enhance the maturation of neurons. We functionalized IKVAV on three artificial ECMs based on supramolecular nanofibers containing peptide amphiphile (PA) molecules. All nanofibers displayed on their surface the same IKVAV signal but differed in the nature of their non-bioactive domains. We found that nanofibers with greater intensity of internal supramolecular motion had enhanced bioactivity toward iPSC-derived motor and cortical neurons. Proteomic, biochemical and functional assays revealed that scaffolds with highly mobile molecules lead to enhanced β 1-integrin pathway activation, reduced aggregation, increased arborization, and mature electrophysiological activity of neurons. Our work resolves long-standing limitations of culturing iPSC-derived neurons, and highlights the importance of designing bioactive ECMs to study the development, function and dysfunction of human neurons in vitro.

Sarah Daoudi is a Field Application Scientist that provides solutions and consultation for Thermo Fisher Scientific’s cell and gene therapy workflows and Gibco Cell Therapy Systems (CTS) brand of products. Sarah has expertise in CAR-T workflows, manufacturing, viral vector production (lentivirus and adeno-associated viruses), and drug development. Formerly a Scientist II at Thermo Fisher Scientific, she helped launch the CTS Rotea System by producing application data, writing and developing new protocols, as well as extensive troubleshooting and customer support. In her previous role as Process Development Research Associate at City of Hope (Duarte, California), Sarah oversaw and worked on improving, troubleshooting, and development of Phase I CAR-T cell therapeutic drugs and workflows for over ten different IRBs. Sarah received her master’s degree in Cell and Molecular Biology from California State University, Fullerton (California).

At the foundation of any cell therapy development and manufacturing workflow, the quality and standardization of the process directly impacts the final product’s viability and the efficacy of patient treatment. Most of initial research into immunotherapies are currently performed using open systems, which can contribute to errors and contamination, resulting in the failure to produce a viable cell therapy. Additionally, donor product variability can lead to differences in cell composition, cell viability, and sensitivity during the expansion stages of the manufacturing. Therefore, cell therapy workflows must be flexible to allow for various modifications, while still yielding a standardized cell product, regardless of the input material harvest from the patient or donor. The ideal isolation workflow would also be automated, closed, and consistent. Researchers are looking for improvements that will decrease contamination, improve batch-to-batch consistency, and allow for monitoring and capture of critical information, which will help with the ever-changing regulatory environment. To this end, cell therapy manufacturers are turning to automated, closed systems with integrated software controls to achieve lower manufacturing costs, maintain product consistency, and meet regulatory requirements. At Thermo Fisher Scientific, we work with you to create a comprehensive process control, digital connection and data stream of mature manufacturer for commercialized products.

Dr. Fossati is a NYSCF Senior Investigator at The NYSCF Research Institute where she focuses on advancing preclinical studies of neurodegenerative and neuroinflammatory disorders, utilizing human iPSC-derived brain cell study. Dr. Fossati received her undergraduate degree in Pharmaceutical Biotechnology and her PhD in Stem Cell Biology from University of Bologna. Dr. Fossati did her postdoctoral training, for which she received the NYSCF Druckenmiller fellowship, in the laboratory of Dr. Hans Willem Snoeck at the Black Family Stem Cell Institute at Mount Sinai and she was recruited in 2011 by the NYSCF Research Institute as principal investigator. Bringing her stem cell expertise, Dr. Fossati has pioneered the development of human stem cell-based models to study the role of glia in neurodegeneration and neuroinflammation. Dr. Fossati established protocols to generate oligodendrocytes, astrocytes, microglia and neuronal cell types and she is developing organoids and co-culture systems to identify and target the key pathogenic mechanisms leading to neurodegeneration and/or demyelination in progressive multiple sclerosis, Alzheimer’s disease and other disorders of the central nervous system.

The human central nervous system (CNS) is a complex organization of several different cell types including neurons, astrocytes, oligodendrocytes, and microglia. While the majority develop from the ectodermal lineage, microglia differentiate from mesodermal progenitors that arise in the yolk sac and migrate to the brain during embryonic development. Our lab focuses on establishing induced pluripotent stem cell (iPSC) models that contain all the major brain cell types to study the cell-cell interactions in the context of neuroinflammation and neurodegeneration. We have developed a protocol for generating CX3CR1+/CD14+ microglia progenitor cells (MGPs), and a protocol for generating oligocortical organoids comprised of cortical neurons, astrocytes and oligodendrocyte lineage cells. We have integrated MGPs within these organoids and showed that they migrate, differentiate into mature microglia, and persist for over six months. Next, we adapted our organoid models to perform long-term cultures in low-Earth orbit (LEO), where the microgravity provides a unique environment to study the biology of neural cells and microglia. Indeed, observations made in both astronauts and animal studies suggest that microgravity not only affect the cardiac, musculoskeletal, and immune systems, but also the CNS, potentially accelerating inflammation, and neurodegeneration. We successfully cultured organoid models with integrated microglia onboard the International Space Station for one month using iPSC lines derived from people with Parkinson’s disease, progressive multiple sclerosis, and age/sex matched controls. Upon return to Earth these organoids showed altered gene expression and protein secretion profiles compared to the ground control samples. These results lay the groundwork for further studies to dissect fundamental neurodegenerative mechanisms and understand the impact of microgravity on disease-relevant processes to develop potential treatments for patients on Earth and countermeasures for astronauts.

Xin is Assistant Professor of Neurosurgery at Boston Children’s Hospital and Harvard Medical School. He received his Ph.D. in neurobiology from Pennsylvania State University, and completed his postdoctoral training with Drs. Rudolf Jaenisch and Mriganka Sur at the Whitehead Institute for Biomedical Research/MIT. Xin’s scientific interest has been focused on understanding the molecular and cellular basis of pediatric brain disorders in order to ultimately develop therapeutics that can be translated to the clinic to improve patient care.

One in six children in the US is afflicted by Neurodevelopmental disorders (NDD). Genetics research in NDD patients have discovered various risk genes in which monoallelic nonsense mutations that truncate and inactivate half of the protein products leads to gene haploinsufficiency, directly causing NDD symptoms. Unfortunately, such discoveries have not been effectively leveraged to support the development of therapeutics that are effective, accessible, and affordable for NDD patients. Our work represents a new avenue for brain disease drug discovery, one that employs small molecule compounds to enhance the gene expression output of the intact wild-type allele of NDD risk genes to alleviate disease symptoms. We will discuss our work that uses gene editing in human stem cells and high-throughput screening to demonstrate the feasibility of repurposing a group of FLT3 kinase inhibitors originally used for treating blood cancer into drugs that effectively alleviate NDD symptoms.

Dr Leitch is an Academic Clinical Lecturer in Genetics at Imperial College London and a Medical Research Council (MRC) Investigator at the MRC London Institute of Medical Sciences where he leads the Germline & Pluripotency group.

Primordial germ cells (PGCs) are the embryonic precursors of the gametes, giving rise to either sperm or eggs during normal development. However, when placed in culture PGCs can undergo conversion to form naïve pluripotent stem cells called embryonic germ (EG) cells. Studying this conversion has led to advances in PGC culture conditions and improved our understanding of how pluripotency is regulated during germline development. It is also now possible to derive PGC-like cells from pluripotent stem cells from a range of mammalian species, including humans. This growing in vitro toolkit offers exciting future possibilities to study the mechanism by which cells transition between germline and pluripotent states, which in turn is of clinical relevance to both germ cell tumourigenesis and the study and treatment of infertility.