Speakers - 24daysofstemcells

Speakers

Information regarding speakers and the final agenda will be updated as we approach the virtual event and subject to change until the final agenda is determined. Please check back often for more details.

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Ivana Barbaric

Group Leader, Centre for Stem Cell Biology, University of Sheffield, UK

Survival of the fittest: The cause and consequences of genetic changes in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have the ability to self-renew indefinitely and differentiate into all types of tissue in the body, providing a potentially unlimited source of differentiated cell types for use in regenerative medicine, disease modelling and drug discovery. The use of hPSCs in these applications will necessitate the maintenance of large numbers of undifferentiated, genetically stable cells. Human PSCs are subject to mutations in vitro and in the presence of selection pressures, the variants with mutations that allow for improved growth outcompete their neighbours and overtake the culture. The commonly observed genetic changes in hPSCs are non-random and involve gains of (parts of) chromosomes 1, 12, 17 and 20, indicating that genes within these regions confer selective advantage to mutant cells. Mutations that arise in hPSCs during in vitro culture can affect their behaviour and confound experimental results. For example, variant cells often show signs of neoplastic progression, including reduced apoptosis, growth-factor independence and higher cloning efficiency. Genetic changes can also affect the propensity of hPSCs to differentiate. Altered patterns of differentiation caused by accrued genetic mutations may significantly affect the use of such cell lines in applications that require the production of differentiated derivatives. Further, the commonly observed mutations in hPSCs are also frequent in human embryonal carcinoma cells, the stem cells of germ cell tumours teratocarcinomas. With hPSC derivatives entering the clinical trials, a possibility that genetic changes may confer malignant properties to hPSCs or their differentiated progeny is a major cause of regulatory concern. In our work we are elucidating the molecular mechanisms that underlie the maintenance of the integrity of the hPSC genome, and how disruption of these mechanisms can lead to undesired genetic changes. We are also studying the functional effects of genetic changes on the behavior of hPSCs in vitro. Investigating the causes and consequences of genetic changes in hPSCs will help inform approaches to minimise their occurrence in hPSC cultures.

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Tenneille Ludwig

Director of the WiCell Stem Cell Bank

Focus on stem cell quality: Improving rigor and reproducibility through characterization

As stem cell scientists, the quality of our research is directly related to the quality of the hPSC materials used. Poor quality cells can impact reproducibility, jeopardize results, waste time, and drain resources. In screening materials submitted to the WiCell Stem Cell Bank, we have identified a substantial and concerning variability in cell quality. Further in-depth analysis of a decade of karyotypic data has allowed us to identify specific areas of recurrent karyotypic instability previously unknown. These results highlight the need for improved testing strategies and standards.

As of this abstract, more than 1600 cell lines have been deposited with the WiCell Stem Cell Bank for banking and characterization by 31 providing laboratories. The vast majority of these cell lines were generated through grant-funded projects as a resource for the larger scientific community, and reportedly screened prior to submission. Various testing strategies were used, and available characterization information was provided to WiCell for reference. To date, nearly 800 of these lines have been independently tested by WiCell for thaw viability, genetic stability (karyotype), identity via short tandem repeat (STR) analysis, sterility (bacteria and fungus), and mycoplasma. Of the hPSC lines examined, more than one-third of WiCell screened cell lines failed this routine quality testing. While there were failures across all tests, the majority of cell lines failed due to unexpected abnormal karyotype.

Stem cell lines deposited with the WiCell Stem Cell Bank are karyotyped internally, and WiCell Characterization additionally performs genetic testing for outside organizations. We performed a retrospective analysis on karyotype data collected over the course of a decade, (including more than 15,000 hPSC cultures). This analysis enabled us to identify striking shifts in relative frequencies of recurrent abnormalities; namely, dramatically increasing rates of chromosomes 1 and 20 gains at the expense of chromosome 12 gains. Additionally, we identified the minimal amplicon for all chromosome 1q gains as chromosomal band (segment) 1q32.1, suggesting that this region harbors the driver gene(s) that give this recurrent aberration its advantage in culture.

Overall, these results show that quality screening strategies in use today are variable, and largely insufficient. Based on this data, we can assume that a substantial percentage of materials used in investigator laboratories have unidentified quality issues that will impact research. The reliability and reproducibility of data gained through experimentation is dependent on maintaining normal, consistent cell cultures. Changes in genetic composition can have dramatic impacts on cell function, and therefore experimental results. This underscores the need for routine testing prior to initiating and following studies, particularly genetic analysis to assure cell line stability.

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Erik McIntire

University of Chicago, USA

Focus on stem cell quality: Improving rigor and reproducibility through characterization

As stem cell scientists, the quality of our research is directly related to the quality of the hPSC materials used. Poor quality cells can impact reproducibility, jeopardize results, waste time, and drain resources. In screening materials submitted to the WiCell Stem Cell Bank, we have identified a substantial and concerning variability in cell quality. Further in-depth analysis of a decade of karyotypic data has allowed us to identify specific areas of recurrent karyotypic instability previously unknown. These results highlight the need for improved testing strategies and standards.

As of this abstract, more than 1600 cell lines have been deposited with the WiCell Stem Cell Bank for banking and characterization by 31 providing laboratories. The vast majority of these cell lines were generated through grant-funded projects as a resource for the larger scientific community, and reportedly screened prior to submission. Various testing strategies were used, and available characterization information was provided to WiCell for reference. To date, nearly 800 of these lines have been independently tested by WiCell for thaw viability, genetic stability (karyotype), identity via short tandem repeat (STR) analysis, sterility (bacteria and fungus), and mycoplasma. Of the hPSC lines examined, more than one-third of WiCell screened cell lines failed this routine quality testing. While there were failures across all tests, the majority of cell lines failed due to unexpected abnormal karyotype.

Stem cell lines deposited with the WiCell Stem Cell Bank are karyotyped internally, and WiCell Characterization additionally performs genetic testing for outside organizations. We performed a retrospective analysis on karyotype data collected over the course of a decade, (including more than 15,000 hPSC cultures). This analysis enabled us to identify striking shifts in relative frequencies of recurrent abnormalities; namely, dramatically increasing rates of chromosomes 1 and 20 gains at the expense of chromosome 12 gains. Additionally, we identified the minimal amplicon for all chromosome 1q gains as chromosomal band (segment) 1q32.1, suggesting that this region harbors the driver gene(s) that give this recurrent aberration its advantage in culture.

Overall, these results show that quality screening strategies in use today are variable, and largely insufficient. Based on this data, we can assume that a substantial percentage of materials used in investigator laboratories have unidentified quality issues that will impact research. The reliability and reproducibility of data gained through experimentation is dependent on maintaining normal, consistent cell cultures. Changes in genetic composition can have dramatic impacts on cell function, and therefore experimental results. This underscores the need for routine testing prior to initiating and following studies, particularly genetic analysis to assure cell line stability.

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Phil Yang

Associate Professor of Medicine, Stanford University School of Medicine

Exosomes and beyond: translation of small molecules and cellular organelles

Pharmacologic therapies have improved survival in heart failure (HF) patients over the past three decades. Despite these pioneering medical therapies, HF is the leading diagnosis of hospital admission associated with high morbidity and mortality. This public health epidemic highlights the need for a development of innovative treatment strategies. Heart failure represents bioenergetic imbalance. The disruption of this cellular balance between energy supply and demand underlies the pathogenesis of HF.

Recent evidence indicates that the stem cells exert their therapeutic action via paracrine mechanisms through microvesicles, which show distinct functionalities in tissue self-repair and -maintenance. These microvesicles ranging from 40 to 400 nm in diameter are formed by endosomal membrane and released into the extracellular space. They contain unique cytoplasmic small molecules, proteins, and cellular organelles that function as intercellular messengers and effectors, controlling a wide spectrum of genetic regulation. Our data demonstrated that the miR cluster and mitochondria contained within these microvesicles underlie the mechanism of action of iPSC-derivatives in repairing the injured and vulnerable heart. These microvesicles have therapeutic potential to protect all the cells in the human body against early death, inflammation, hypertrophy, fibrosis, and energy demand. Our data provide unique insights into the potential of endogenous microvesicles generated from the autologous iPSC-derivatives for personalized, precision medicine. This approach, employing a person’s rejuvenated cellular components and molecules from iPSCs, may underlie a novel strategy to address HF.

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Lay Teng Ang

Siebel Investigator and Instructor, Stanford

Turning human pluripotent stem cells into highly-pure and engraftable tissue progenitors

Our overall goal is to understand the mechanisms through which stem cells differentiate into progressively-specialized cell-types and to harness this knowledge to artificially generate pure populations of desired cell-types from stem cells. Pluripotent stem cells (PSCs, which include embryonic and induced pluripotent stem cells) have the remarkable ability to generate any of the hundreds of diverse cell-types in the body. However, it has been notoriously difficult to guide PSCs to differentiate into a pure population of a given cell-type. Current differentiation strategies typically generate heterogeneous cell populations unsuitable for basic research or clinical applications. To address this challenge, we mapped the cascade of branching lineage choices through which PSCs differentiate into a variety of endodermal and mesodermal cell-types. We further developed effective methods to differentiate PSCs into specific lineages by providing the extracellular signal(s) that specify a given lineage while inhibiting the signals that induce the alternate fate(s), enabling the generation of highly-pure human heart, bone and liver progenitors from PSCs. The human tissue progenitors are engraftable and could form tissues in mice. The ability to efficiently generate large numbers of such progenitors in vitro may have important ramifications for regenerative medicine.


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Hassan Rashidi

UCL, Great Ormond Street Institute of Child Health

Human Pluripotent Stem cells-derived 3D Hepatospheres

Liver disease is the fifth most common cause of death in the UK and the death toll is rising. Liver transplantation is an effective procedure to treat end-stage liver disease and organ failure, however donor organ shortage represents a significant problem. Therefore, there is a clear imperative to develop novel and scalable alternatives to treat human liver disease. The use of the major metabolic cell type of the liver, the hepatocyte, as a cell-based therapy to treat human metabolic liver disease has proved successful [1]. However, like the whole organ itself, primary human hepatocytes are a limited resource with which to tackle the worldwide issue of liver disease.

The pluripotent stem cells can provide a credible alternative source to generate quality assured human liver tissue for the clinic. To this end, several protocols have been developed to efficiently generate hepatocyte-like cells (HLCs), mainly employing two-dimensional differentiation systems [2-8]. Despite recent improvements [9-11], 2D-derived HLCs exhibit foetal features and transient phenotype in vitro, limiting their clinical application. Efforts to overcome these limiting factors have led to the building of three dimensional (3D) liver organoids [12]. However, they are not suitable for clinical application due to their reliance on animal-derived and undefined biological components [13-17].

Our studies focused on the development of 3D hepatospheres under serum-free and GMP-ready conditions. Notably, generated 3D tissues exhibited stable liver phenotype for over 365 days in culture and provided critical liver support in tyrosinemia type-I animal models. We believe that our study delivers a blue-print to effectively treat certain liver diseases in the future.


1. Alwahsh, S.M., H. Rashidi, and D.C. Hay, Liver cell therapy: is this the end of the beginning? Cell Mol Life Sci, 2017.

2. Hay, D.C., et al., Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities. Cloning Stem Cells, 2007. 9(1): p. 51-62.

3. Hay, D.C., et al., Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo. Stem Cells, 2008. 26(4): p. 894-902.

4. Hay, D.C., et al., Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling. Proc Natl Acad Sci U S A, 2008. 105(34): p. 12301-6.

5. Agarwal, S., K.L. Holton, and R. Lanza, Efficient differentiation of functional hepatocytes from human embryonic stem cells. Stem Cells, 2008. 26(5): p. 1117-27.

6. Sullivan, G.J., et al., Generation of functional human hepatic endoderm from human induced pluripotent stem cells. Hepatology, 2010. 51(1): p. 329-35.

7. Hannan, N.R., et al., Production of hepatocyte-like cells from human pluripotent stem cells. Nat Protoc, 2013. 8(2): p. 430-7.

8. Loh, K.M., et al., Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations. Cell Stem Cell, 2014. 14(2): p. 237-52.

9. Takayama, K., et al., Long-term self-renewal of human ES/iPS-derived hepatoblast-like cells on human laminin 111-coated dishes. Stem Cell Reports, 2013. 1(4): p. 322-35.

10. Cameron, K., et al., Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes. Stem Cell Reports, 2015. 5(6): p. 1250-1262.

11. Wang, Y., et al., Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells. J Vis Exp, 2017(121).

12. Szkolnicka, D. and D.C. Hay, Concise Review: Advances in Generating Hepatocytes from Pluripotent Stem Cells for Translational Medicine. Stem Cells, 2016. 34(6): p. 1421-1426.

13. Gieseck, R.L., 3rd, et al., Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture. PLoS One, 2014. 9(1): p. e86372.

14. Takebe, T., et al., Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nat Protoc, 2014. 9(2): p. 396-409.

15. Camp, J.G., et al., Multilineage communication regulates human liver bud development from pluripotency. Nature, 2017. 546(7659): p. 533-+.

16. Huch, M., et al., In vitro expansion of single Lgr5(+) liver stem cells induced by Wnt-driven regeneration. Nature, 2013. 494(7436): p. 247-250.

17. Huch, M., et al., Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver. Cell, 2015. 160(1-2): p. 299-312.

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Ting Zhou

Memorial Sloan Kettering Cancer Center

Using human pluripotent stem cells as platform for disease investigation and drug discovery

Human pluripotent stem cells (hPSCs) and the functional cells derived from them can be utilized for chemical screen, complex disease investigation and drug discovery. The first example I present today is to set up a novel hPSC-based platform to uncover potential gene-environment interactions. We have performed an environment chemical screen on hESC derived β-like cells, and found that a commonly used pesticide, propargite, induced pancreatic β-cell death, a pathological hallmark of diabetes which is impacted by both genetic and environmental factors.  We then assessed the potential gene-environment interactions using isogenic hPSC lines for genetic variants associated with diabetes, and found a that GSTT1−/− pancreatic β-like cells were hypersensitive to propargite-induced cell death. This study identified an environmental chemical that could contribute to human β-cell loss and validated the hPSC-based platform for determining gene-environment interactions. The second example is to utilize hPSC-derived neural progenitors to perform a high content screen for anti-ZIKV drug discovery. Using the platform, we have screened >1,000 FDA-approved drug candidates, and found two with anti-ZIKV activity: Hippeastrine hydrobromide (HH) and Amodiaquine dihydrochloride dihydrate (AQ). The drug candidates were further validated with hPSC-derived forebrain organoids in vitro and a murine model in vivo.

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Indrani Datta

NI of Mental Health and Neurosciences, Bengaluru, India

Parkinson’s Disease in a Petri Dish: Harnessing iPSCs to develop an improved model of Personalized Medicine

Parkinson’s disease (PD) is multifactorial and clinically heterogeneous. Recent advances in research in PD increasingly indicate that genes and environmental factors can interact to modulate the risk of disease. Indeed, epidemiological reports suggest distinct differences in gene variants in PD between western and eastern populations. Thus far, differences in PD-related gene variants between East and West have been reported for LRRK2, MAPT, BST1 and PARK16 genes. These differences can cause phenotypic alterations in pathobiologies and drug metabolic pathways, and it is thus crucial that PD treatment strategies take these ethnicity variations into consideration. However, current disease modelling and drug development programs largely use animal models that fail to replicate human ethnicity differences. It is now well-established that humans and mice have considerable developmental, genetic and physiological differences, and that genetic mutations for human PD do not completely replicate the disease phenotype in mice. Therefore, the search for models that mirror patient-specific PD phenotypic manifestations as closely as possible still continues. Patient-derived human induced pluripotent stem cells (iPSCs) can fill this lacuna between genetic association studies and underlying molecular mechanisms. iPSCs can be selectively differentiated into specialized cells of all three lineages, and thus PD patient-specific iPSCs can be used not only to derive dopaminergic neurons but also intestinal cells and microglia which are also known to be affected under certain familial PD conditions. The potential to derive other specialized CNS cells from iPSCs also open the gates to test any drug-induced effect on these cells, and will help to establish specific genetic risk factors to assess genetic sub-populations’ differing responses to treatment. Unlike other artificially-induced models, endogenous cellular machinery and transcriptional feedback are preserved in iPSC models, a fundamental step in accurately modelling this genetically complex disease. The underlying cellular pathogenesis can be accurately traced and modelled using these cells, which also hold the potential for mutations to be genetically corrected and their phenotypic manifestation on the disease pathology evaluated. Thus, iPSC derived cells can be harnessed for developing improved cellular models which will be instrumental in dissecting a complex pathological process into simpler molecular events; in turn a step forward towards personalized medicine in PD.

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Filomena Pirozzi

Research Scientist III, Seattle's Children Research Institute

Using iPSCs and Cerebral organoids to dissect the pathophysiology of brain growth disorders

Filomena Pirozzi1, Gaia Ruggeri1, Matthew Berkseth1, Anthony Wynshaw-Boris2, William B. Dobyns1, 2, Ghayda M. Mirzaa1,2

1) Center for integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA
2) Department of Genetics and Genome Sciences, Case Western Reserve university, Cleveland, OH
3) Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, WA

Microcephaly (MIC) and Megalencephaly (MEG) are neurodevelopmental disorders at the opposite ends of the brain growth spectrum, with severe MIC defined as occipitofrontal circumference (OFC) at least 3 standard deviation (SD) below the mean, and MEG presenting with OFC > 3SD above the mean. MIC and MEG are characterized by variable brain and cortical abnormalities. This spectrum is associated with significant pediatric morbidity and mortality including epilepsy, autism and intellectual disability. In this talk, I will present two examples of how our team has used iPSCs and cerebral organoids to model in vitro these two phenotypes, with the aim to identify the molecular cascade of events and, possibly, new drug targets. As example of MIC, we focused on LIG4 Syndrome, a rare autosomal disorder leading to deficiency of the DNA repair pathway Non-Homologous End Joining (NHEJ). In addition, we selected the PI3K-MTOR pathway as example of MEG, as these genes have been identified to cause Focal Cortical Displasya (FCD), the most common cause of pediatric epilepsy. We generated induced pluripotent stem cells (iPSCs) carrying the common LIG4R278H, PIK3CAH1047R or MTORT1977I mutations. We differentiated the obtained iPSCs into Neuronal Progenitors (NPCs), cortical neurons, and cerebral organoids and performed functional assays including population doubling time, senescence, analysis of proliferation and apoptosis. We were able to recapitulate the microcephaly and megalencephaly phenotypes in vitro, identifying different molecular events leading to the pathogenesis of these phenotypes for each gene. Interestingly, despite sharing similar clinical features and being nodes of the same pathways, we revealed both overlapping and exclusive cellular phenotypes in PIK3CA and MTOR mutant cell lines, indicating that classifying MEG based on the pathway might not be enough to discriminate among different pathogenesis. These results, together with previous work from other labs, provide proof-of-concept that iPSC and cerebral organoids are ideal tools to dissect the pathophysiology of brain growth disorders in humans. Our future directions is to use these tools in order to perform high-throughput drug screening in order to identify novel therapeutic candidates.

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Michael Nestor

Hussman Institute

Characterization of multiple phenotypes in individuals with idiopathic autism using high-throughput screening of hiPSC-derived cortical organoids 

Individuals with Autism Spectrum Condition (ASC) exhibit a number of neurodevelopmental and behavioral challenges that are centered on repetitive behaviors, interests, and difficulties with social and emotional communication. These behaviors may have their basis in aberrant neurodevelopmental mechanisms in cortical networks conferred by a convergence of genes.  Although critical, gaining an understanding of phenotypic differences inherent in the neurons of autistic individuals remains a central challenge for the field. An emergent hypothesis in autism research is that the imbalance of excitatory and inhibitory (E/I) inputs within neural networks in autistic brains may account for some of the behavioral phenotypes observed in these individuals. The homeostatic regulation of the balance between E/I neurons and their synaptic inputs is required to maintain the narrow range of optimal neuronal spiking required for the transfer of information within the brain.  

To understand the role of E/I inputs in autism we employ a human induced pluripotent stem cell (hiPSC)-derived 3D organoid model system called a serum free embryoid body (SFEB). This system is an in vitro platform that may more accurately recapitulate human cortical development. SFEBs are used to test for potential differences in the morphology and network-level function that are specific to cortical neurons derived from ASC patient hiPSCs. We combine the use of SFEBs with high-throughput approaches to compensate for heterogeneity and variability inherent in 3D cultures. High-throughput screening using the ThermoFisher ArrayScan XTi platform was employed to quantify GABA+ and VGLUT+ cells in SFEBs and VGLUT+ neuron morphology in individuals with and without autism. Network-level activity was recorded from SFEBs using multi-electrode arrays. We observed fewer GABA+ cells and more spiking in a number of individuals with autism within our small cohort. These findings indicate that potential E/I deficits found in this ASC cohort can be detected using high-throughput approaches. 

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Edwin Golez

Thermo Fisher Scientific

3D Neural Organoid Formation from iPSCs

Advances in cell culture techniques have focused on creating 3-dimensional (3D) systems in an attempt to represent in vivo cell–cell relationships and microenvironments in vitro utilizing adult and pluripotent stem cells (ASCs and PSCs). Starting with a PSC culture, we aggregate the cells to form an embryoid body (EB), when given the proper cues, will undergo self-differentiation and morphogenesis that better recapitulates the in vivo cell to cell interactions and microanatomy of a given tissue type. The development of a 3D organ-like (organoid) system requires the application of growth factors, small molecules, and other media supplements to guide the formation of organoid systems based on the embryogenesis and adult stem cell biology principles. In this training video, we will show you how to develop 3D neural organoids from iPSC culture utilizing a feeder-free system, our Gibco Geltrex matrix, and Thermo Nunclon Sphera microplates.

Cancer Spheroid Formation Using Nunclon Sphera Microplates

2D cultured cells can differ in terms of both physiology and cellular responses compared with cells in vivo. Mounting evidence suggests that culturing cells in 3D is more representative of the in vivo environment, creating more physiological cell models. Spheroids, or sphere cultures, have become especially popular area of 3D in vitro culture due to there great potential for use in studies that investigate growth and function of both normal and malignant tissues. Spheroids offer particular benefits in cancer biology, where they contribute immense value in examining the growth and behavior of tumors since they share several key histomorphological and functional traits that include the formation of cell-cell contacts, decreased proliferation, increased survival rates, and a hypoxic core. In this training video, we will demonstrate how to create 3D cancer spheroids using Nunclon Sphera 96 well U-bottom microplates.

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Gang Li

The Chinese University of Hong Kong

Circulating Mesenchymal Stem Cells in Bone Healing 

Mesenchymal stem cells (MSCs) have been found in cord blood and peripheral blood (PB) of mammalian species including human, guinea pig, mice, rat, dog, horse and rabbit.  The number of MSCs in PB (PB-MSCs) is rare and their biological role was not fully defined.  We have found increased numbers of circulating MSCs in peripheral blood in patients with long bone fracture, non-union and in patients with cancers.  The number of PB-MSCs was approximately 9 times higher in the cancer patients, suggesting there is systemic recruitment of MSCs during cancer development.  We have compared the difference between the circulating MSCs and bone marrow derived MSCs and found that they share similar phenotype in vitro, but the gene expression profile between the two cell populations was significantly different.  cDNA microarray analysis and quantitative RT-PCR confirmed 10 genes that are differentially expressed with more than 10 folds difference, such as  cellular retinol-binding protein 1 (CRBP1), cadherin 2, bone morphogenetic protein 6 (BMP6), SRY-box containing gene 11 (Sox11), the aquaporin 1 (AQP1), et al.  These genes are now being further investigated for their role in MSCs migration, homing and multiple-differentiation potential.  In terms of potential clinical implications of PB-MSCs, we have demonstrated that allogenic PB-MSCs enhanced bone regeneration in rabbit ulna critical-sized bone defect model. We also demonstrated that BM-MSCs can be recruited via circulation toward the sites of bone fracture and participate fracture healing in rabbits.  We have also demonstrated that systemically administrated allogenic MSCs could home to fracture sites and promote fracture healing.  In conclusion, PB-MSCs are new cell source of cells that may play very important roles in development, repair and disease progression.

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Abigail Harris Becker

Thermo Fisher Scientific

CTS StemPro HSC Expansion Medium for Cell and Gene Therapies

A major limitation in the ex vivo expansion of harvested human hematopoietic stem-progenitor cells (HSPCs) is the rapid differentiation of HSPCs at the expense of the most primitive pluripotent hematopoietic stem cells (HSCs). A culture system that expands both short-term progenitor cells and long-term repopulating HSCs could benefit translational research and clinical applications such as HSC transplantation and gene therapies. To address this challenge, we developed CTS StemPro HSC Expansion Medium, a xeno-free, serum-free medium designed with regulatory compliance in mind.  Using a Design of Experiments approach, media constituents were systematically modified and each iteration was evaluated with the goal to maximize ex vivo expansion of hematopoietic stem-progenitor cell (HSPC) immunophenotypes.

CTS StemPro HSC Expansion Medium supplemented with FLT3L, SCF (also known as KITL), TPO, IL-3, and IL-6 (“FST36”) expands human CD34+ cells from mobilized peripheral blood, cord blood, and bone marrow. For example, primary human CD34+ cells from mobilized peripheral blood cultured for 7 days in FST36-containing CTS StemPro™ HSC Expansion Medium resulted in a ~100-fold increase in the number of CD34+CD45+Lin- cells and a ~2000-fold increase in the number of CD34+Lin-CD90+CD45RA- cells (an early HSPC immunophenotype), compared to uncultured cells. Expanded CD34+ cells maintained key hematopoietic stem cell characteristics including in vitro differentiation into erythroid and non-erythroid cell colonies and expression of high levels of aldehyde dehydrogenase. Importantly, in vivo transplantation studies indicate that the expanded CD34+ cells can engraft into the bone marrow of immuno-deficient mice. In addition, utilizing liposome-mediated electroporation to deliver Cas9 RNPs (500 ng 1.4 kb GFP), we observed indel rates of up to 60% and a knock-in rate of 10% in cultured CD34+ cells. Both cultured CD34+ cells and PBMCs demonstrated a high reprogramming efficiency (~0.3%) with the CytoTune-iPS Sendai Reprogramming Kit. We are enthusiastic to introduce CTS StemPro HSC Expansion Medium, which enables a complete xeno-free and serum-free workflow for the utilization of human CD34+ cells in gene and cell therapies.

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Ben Fryer

Pluristyx

Manufacturing pluripotent cell therapeutics: the importance of quality from the very beginning 

Unlike many other forms of cell therapeutics that are tailored for individual patients and rely on small batch production, pluripotent cell therapies for both allogeneic and autologous use have as one of the first steps the establishment of a master cell bank that must last the lifetime of the product. Thus, raw material quality is paramount from the beginning of the development process.  Obtaining high-quality raw materials from suppliers experienced in supporting cell therapy development—from manufacturing to delivery—can increase the probability of success and head off costly surprises that could cause an untimely demise for a promising pluripotent cell therapy candidate.


Short biography:

Ben Fryer is the CEO and co-founder of Pluristyx Inc. providing GMP pre-expanded pluripotent stem cells, contract development services, and consultancy for groups and company developing cell and tissue therapies.  Ben has almost 25 years of experience as a leader and scientist in large pharma, academia, and start-up companies developing combination products and cell therapies. He has worked to generate GMP/ clinical grade pluripotent stem cells from early banking through large scale expansion using scalable, closed-loop, suspension reactor processes to manufacture cell therapy treatments for diabetes and heart disease. 

Ben is an inventor on multiple issued patents and patent applications for BetaLogics/Janssen/ J&J, including a bioreactor based, stem cell suspension expansion and differentiation process and a product currently marketed by Thermo-Fisher(TM) as Nunclon-Vita. 

Ben earned his BA from Colorado College, his PhD from UPENN, and studied as a post-doctoral fellow in the Howard Hughes Laboratory of M. Celeste Simon at the University of Pennsylvania investigating the role of Hypoxia Inducible Factor in cancer and stem cell development.