Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells.

Efficient translation of human induced pluripotent stem cells (hiPSCs) requires scalable cell manufacturing strategies for optimal self-renewal and functional differentiation. Traditional manual cell culture is variable and labor intensive, posing challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient- and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation and generated functional neurons, cardiomyocytes, and hepatocytes. To validate our approach, we compared robotic and manual cell culture operations and performed comprehensive molecular and cellular characterizations (e.g., single-cell transcriptomics, mass cytometry, metabolism, electrophysiology) to benchmark industrial-scale cell culture operations toward building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy.

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

Efficient translation of human induced pluripotent stem cells (hiPSCs) depends on implementing scalable cell manufacturing strategies that ensure optimal self-renewal and functional differentiation. Currently, manual culture of hiPSCs is highly variable and labor-intensive posing significant challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This streamlined approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient-and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation to generate primary embryonic germ layers and more mature phenotypes such as neurons, cardiomyocytes, and hepatocytes. To validate our approach, we carefully compared robotic and manual cell culture and performed molecular and functional cell characterizations (e.g. bulk culture and single-cell transcriptomics, mass cytometry, metabolism, electrophysiology, Zika virus experiments) in order to benchmark industrial-scale cell culture operations towards building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy. Combining stem cell-based models and non-stop robotic cell culture may become a powerful strategy to increase scientific rigor and productivity, which are particularly important during public health emergencies (e.g. opioid crisis, COVID-19 pandemic).

Chemically Defined Neural Conversion of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) are characterized by their ability to self-renew and differentiate into any cell type of the human body. To fully utilize the potential of hPSCs for translational research and clinical applications, it is critical to develop rigorous cell differentiation protocols under feeder-free conditions that are efficient, reproducible, and scalable for high-throughput projects. Focusing on neural conversion of hPSCs, here we describe robust small molecule-based procedures that generate neural stem cells (NSCs) in less than a week under chemically defined conditions. These protocols can be used to dissect the mechanisms of neural lineage entry and to further develop systematic protocols that produce the cellular diversity of the central nervous system at industrial scale.

A Drug Screen using Human iPSC-Derived Hepatocyte-like Cells Reveals Cardiac Glycosides as a Potential Treatment for Hypercholesterolemia.

Efforts to identify pharmaceuticals to treat heritable metabolic liver diseases have been hampered by the lack of models. However, cells with hepatocyte characteristics can be produced from induced pluripotent stem cells (iPSCs). Here, we have used hepatocyte-like cells generated from homozygous familial hypercholesterolemia (hoFH) iPSCs to identify drugs that can potentially be repurposed to lower serum LDL-C. We found that cardiac glycosides reduce the production of apolipoprotein B (apoB) from human hepatocytes in culture and the serum of avatar mice harboring humanized livers. The drugs act by increasing the turnover of apoB protein. Analyses of patient medical records revealed that the treatment of patients with cardiac glycosides reduced serum LDL-C levels. These studies highlight the effectiveness of using iPSCs to screen for potential treatments for inborn errors of hepatic metabolism and suggest that cardiac glycosides could provide an approach for reducing hepatocyte production of apoB and treating hypercholesterolemia.

N-glycoprotein surfaceome of human induced pluripotent stem cell derived hepatic endoderm.

Using cell surface capture technology, the cell surface N-glycoproteome of human-induced pluripotent stem cell derived hepatic endoderm cells was assessed. Altogether, 395 cell surface N-glycoproteins were identified, represented by 1273 N-glycopeptides. This study identified N-glycoproteins that are not predicted to be localized to the cell surface and provides experimental data that assist in resolving ambiguous or incorrectly annotated transmembrane topology annotations. In a proof-of-concept analysis, combining these data with other cell surface proteome datasets is useful for identifying potentially cell type and lineage restricted markers and drug targets to advance the use of stem cell technologies for mechanistic developmental studies, disease modeling, drug discovery, and regenerative medicine.

Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

FGF2 mediates hepatic progenitor cell formation during human pluripotent stem cell differentiation by inducing the WNT antagonist NKD1.

Fibroblast growth factors (FGFs) are required to specify hepatic fate within the definitive endoderm through activation of the FGF receptors (FGFRs). While the signaling pathways involved in hepatic specification are well understood, the mechanisms through which FGFs induce hepatic character within the endoderm are ill defined. Here we report the identification of genes whose expression is directly regulated by FGFR activity during the transition from endoderm to hepatic progenitor cell. The FGFR immediate early genes that were identified include those encoding transcription factors, growth factors, and signaling molecules. One of these immediate early genes encodes naked cuticle homolog 1 (NKD1), which is a repressor of canonical WNT (wingless-type MMTV integration site) signaling. We show that loss of NKD1 suppresses the formation of hepatic progenitor cells from human induced pluripotent stem cells and that this phenotype can be rescued by using a pharmacological antagonist of canonical WNT signaling. We conclude that FGF specifies hepatic fate at least in large part by inducing expression of NKD1 to transiently suppress the canonical WNT pathway.

Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells.

Maintenance and differentiation of human pluripotent stem cells (hPSCs) usually requires culture on a substrate for cell adhesion. A commonly used substratum is Matrigel purified from Engelbreth-Holm-Swarm sarcoma cells, and consists of a complex mixture of extracellular matrix proteins, proteoglycans, and growth factors. Several studies have successfully induced differentiation of hepatocyte-like cells from hPSCs. However, most of these studies have used Matrigel as a cell adhesion substrate, which is not a defined culture condition. In an attempt to generate a substratum that supports undifferentiated properties and differentiation into hepatic lineage cells, we designed novel substrates consisting of vitronectin fragments fused to the IgG Fc domain. hPSCs adhered to these substrates via interactions between integrins and the RGD (Arg-Gly-Asp) motif, and the cells maintained their undifferentiated phenotypes. Using a previously established differentiation protocol, hPSCs were efficiently differentiated into mesendodermal and hepatic lineage cells on a vitronectin fragment-containing substrate. We found that full-length vitronectin did not support stable cell adhesion during the specification stage. Furthermore, the vitronectin fragment with the minimal RGD-containing domain was sufficient for differentiation of human induced pluripotent stem cells into hepatic lineage cells under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs.

Aneuploidy is permissive for hepatocyte-like cell differentiation from human induced pluripotent stem cells.

BACKGROUND: The characterization of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) routinely includes analyses of chromosomal integrity. The belief is that pluripotent stem cells best suited to the generation of differentiated derivatives should display a euploid karyotype; although, this does not appear to have been formally tested. While aneuploidy is commonly associated with cell transformation, several types of somatic cells, including hepatocytes, are frequently aneuploid and variation in chromosomal content does not contribute to a transformed phenotype. This insight has led to the proposal that dynamic changes in the chromosomal environment may be important to establish genetic diversity within the hepatocyte population and such diversity may facilitate an adaptive response by the liver to various insults. Such a positive contribution of aneuploidy to liver function raises the possibility that, in contrast to existing dogma, aneuploid iPSCs may be capable of generating hepatocyte-like cells that display hepatic activities. RESULTS: We examined whether a human iPSC line that had multiple chromosomal aberrations was competent to differentiate into hepatocytes and found that loss of normal chromosomal content had little impact on the production of hepatocyte-like cells from iPSCs. CONCLUSIONS: iPSCs that harbor an abnormal chromosomal content retain the capacity to generate hepatocyte-like cells with high efficiency.

Differentiation of hepatocytes from pluripotent stem cells.

Differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells into hepatocyte-like cells provides a platform to study the molecular basis of human hepatocyte differentiation, to develop cell culture models of liver disease, and to potentially provide hepatocytes for treatment of end-stage liver disease. Additionally, hepatocyte-like cells generated from human pluripotent stem cells could serve as platforms for drug discovery, determination of pharmaceutical-induced hepatotoxicity, and evaluation of idiosyncratic drug-drug interactions. Here, we describe a step-wise protocol previously developed in our laboratory that facilitates the highly efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte-like cells. Our protocol uses defined culture conditions and closely recapitulates key developmental events that are found to occur during hepatogenesis.

Musashi2 is required for the self-renewal and pluripotency of embryonic stem cells.

Recent studies have shown that the RNA binding protein Musashi 2 (Msi2) plays important roles during development. Msi2 has also been shown to be elevated in several leukemias and its elevated expression has been linked with poorer prognosis in these cancers. Additionally, in embryonic stem cells (ESC) undergoing the early stages of differentiation, Msi2 has been shown to associate with the transcription factor Sox2, which is required for the self-renewal of ESC. These findings led us to examine the effects of Msi2 on the behavior of ESC. We determined that ESC express two isoforms of Msi2, the larger canonical isoform (isoform 1) and a shorter, splice-variant isoform (isoform 2). Using multiple shRNA lentiviral vectors, we determined that knockdown of Msi2 disrupts the self-renewal of ESC and promotes their differentiation into cells that express markers associated with mesoderm, ectoderm, and trophectoderm. Moreover, our studies indicate that the extent of differentiation and the loss of self-renewal capacity correlate with the levels to which Msi2 levels were decreased. We extended these findings by engineering ESC to inducibly express either Msi2 isoform1 or isoform 2. We determined that ectopic expression of Msi2 isoform 1, but not isoform 2, enhances the cloning efficiency of ESC. In addition, we examined how Msi2 isoform 1 and isoform 2 affect the differentiation of ESC. Interestingly, ectopic expression of either Msi2 isoform 1 or isoform 2 does not affect the pattern of differentiation induced by retinoic acid. Finally, we show that ectopic expression of either isoform 1 or isoform 2 is not sufficient to block the differentiation that results from the knockdown of both isoforms of Msi2. Thus, it appears that both isoforms of Msi2 are required for the self-renewal of ESC.

Determination of protein interactome of transcription factor Sox2 in embryonic stem cells engineered for inducible expression of four reprogramming factors.

Unbiased proteomic screens provide a powerful tool for defining protein-protein interaction networks. Previous studies employed multidimensional protein identification technology to identify the Sox2-interactome in embryonic stem cells (ESC) undergoing differentiation in response to a small increase in the expression of epitope-tagged Sox2. Thus far the Sox2-interactome in ESC has not been determined. To identify the Sox2-interactome in ESC, we engineered ESC for inducible expression of different combinations of epitope-tagged Sox2 along with Oct4, Klf4, and c-Myc. Epitope-tagged Sox2 was used to circumvent the lack of suitable Sox2 antibodies needed to perform an unbiased proteomic screen of Sox2-associated proteins. Although i-OS-ESC differentiate when both Oct4 and Sox2 are elevated, i-OSKM-ESC do not differentiate even when the levels of the four transcription factors are coordinately elevated ∼2-3-fold. Our findings with i-OS-ESC and i-OSKM-ESC provide new insights into the reasons why ESC undergo differentiation when Sox2 and Oct4 are elevated in ESC. Importantly, the use of i-OSKM-ESC enabled us to identify the Sox2-interactome in undifferentiated ESC. Using multidimensional protein identification technology, we identified >70 proteins that associate with Sox2 in ESC. We extended these findings by testing the function of the Sox2-assoicated protein Smarcd1 and demonstrate that knockdown of Smarcd1 disrupts the self-renewal of ESC and induces their differentiation. Together, our work provides the first description of the Sox2-interactome in ESC and indicates that Sox2 along with other master regulators is part of a highly integrated protein-protein interaction landscape in ESC.

Systems biology provides new insights into the molecular mechanisms that control the fate of embryonic stem cells.

During the last 5 years there has been enormous progress in developing a deeper understanding of the molecular mechanisms that control the self-renewal and pluripotency of embryonic stem cells (ESC). Early progress resulted from studying individual transcription factors and signaling pathways. Unexpectedly, these studies demonstrated that small changes in the levels of master regulators, such as Oct4 and Sox2, promote the differentiation of ESC. More recently, impressive progress has been made using technologies that provide a global view of the signaling pathways and the gene regulatory networks that control the fate of ESC. This review provides an overview of the progress made using several different high-throughput technologies and focuses on proteomic studies, which provide the first glimpse of the protein-protein interaction networks used by ESC. The latter studies indicate that transcription factors required for the self-renewal of ESC are part of a large, highly integrated protein-protein interaction landscape, which helps explain why the levels of master regulators need to be regulated precisely in ESC.

Banf1 is required to maintain the self-renewal of both mouse and human embryonic stem cells.

Self-renewal is a complex biological process necessary for maintaining the pluripotency of embryonic stem cells (ESCs). Recent studies have used global proteomic techniques to identify proteins that associate with the master regulators Oct4, Nanog and Sox2 in ESCs or in ESCs during the early stages of differentiation. Through an unbiased proteomic screen, Banf1 was identified as a Sox2-associated protein. Banf1 has been shown to be essential for worm and fly development but, until now, its role in mammalian development and ESCs has not been explored. In this study, we examined the effect of knocking down Banf1 on ESCs. We demonstrate that the knockdown of Banf1 promotes the differentiation of mouse ESCs and decreases the survival of both mouse and human ESCs. For mouse ESCs, we demonstrate that knocking down Banf1 promotes their differentiation into cells that exhibit markers primarily associated with mesoderm and trophectoderm. Interestingly, knockdown of Banf1 disrupts the survival of human ESCs without significantly reducing the expression levels of the master regulators Sox2, Oct4 and Nanog or inducing the expression of markers of differentiation. Furthermore, we determined that the knockdown of Banf1 alters the cell cycle distribution of both human and mouse ESCs by causing an uncharacteristic increase in the proportion of cells in the G2-M phase of the cell cycle.

Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery.

The rapid formation of numerous tissues during development is highly dependent on the swift activation of key developmental regulators. Recent studies indicate that many key regulatory genes are repressed in embryonic stem cells (ESCs), yet poised for rapid activation due to the presence of both activating (H3K4 trimethylation) and repressive (H3K27 trimethylation) histone modifications (bivalent genes). However, little is known about bivalent gene regulation. In this study, we investigated the regulation of the bivalent gene Sox21, which is activated rapidly when ESCs differentiate in response to increases in Sox2. Chromatin immunoprecipitation demonstrated that prior to differentiation, the Sox21 gene is bound by a complex array of repressive and activating transcriptional machinery. Upon activation, all identified repressive machinery and histone modifications associated with the gene are lost, but the activating modifications and transcriptional machinery are retained. Notably, these changes do not occur when ESCs differentiate in response to retinoic acid. Moreover, ESCs lacking a functional PRC2 complex fail to activate this gene, apparently due to its association with other repressive complexes. Together, these findings suggest that bivalent genes, such as Sox21, are silenced by a complex set of redundant repressive machinery, which exit rapidly in response to appropriate differentiation signals.

Sox2 uses multiple domains to associate with proteins present in Sox2-protein complexes.

Master regulators, such as Sox2, Oct4 and Nanog, control complex gene networks necessary for the self-renewal and pluripotency of embryonic stem cells (ESC). These master regulators associate with co-activators and co-repressors to precisely control their gene targets. Recent studies using proteomic analysis have identified a large, diverse group of co-activators and co-repressors that associate with master regulators, including Sox2. In this report, we examined the size distribution of nuclear protein complexes containing Sox2 and its associated proteins HDAC1, Sall4 and Lin28. Interestingly, we determined that Sox2 and HDAC1 associate with protein complexes that vary greatly in size; whereas, Lin28 primarily associates with smaller complexes, and Sall4 primarily associates with larger complexes. Additionally, we examined the domains of Sox2 necessary to mediate its association with its partner proteins Sall4, HDAC1 and HDAC2. We determined that Sox2 uses multiple and distinct domains to associate with its partner proteins. We also examined the domains of Sox2 necessary to mediate its self-association, and we determined that Sox2 self-association is mediated through multiple domains. Collectively, these studies provide novel insights into how Sox2 is able to associate with a wide array of nuclear proteins that control gene transcription.

Proteomic analysis of Sox2-associated proteins during early stages of mouse embryonic stem cell differentiation identifies Sox21 as a novel regulator of stem cell fate.

Small increases in the levels of master regulators, such as Sox2, in embryonic stem cells (ESC) have been shown to promote their differentiation. However, the mechanism by which Sox2 controls the fate of ESC is poorly understood. In this study, we employed multidimensional protein identification technology and identified >60 nuclear proteins that associate with Sox2 early during ESC differentiation. Gene ontology analysis of Sox2-associated proteins indicates that they participate in a wide range of processes. Equally important, a significant number of the Sox2-associated proteins identified in this study have been shown previously to interact with Oct4, Nanog, Sall4, and Essrb. Moreover, we examined the impact of manipulating the expression of a Sox2-associated protein on the fate of ESC. Using ESC engineered for inducible expression of Sox21, we show that ectopic expression of Sox21 in ESC induces their differentiation into specific cell types, including those that express markers representative of neurectoderm and heart development. Collectively, these studies provide new insights into the range of molecular processes through which Sox2 is likely to influence the fate of ESC and provide further support for the conclusion that the expression of Sox proteins in ESC must be precisely regulated. Importantly, our studies also argue that Sox2, along with other pluripotency-associated transcription factors, is woven into highly interconnected regulatory networks that function at several levels to control the fate of ESC.

Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells.

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression. These small, non-coding RNAs are believed to regulate more than a third of all protein coding genes, and they have been implicated in the control of virtually all biological processes, including the biology of stem cells. The essential roles of miRNAs in the control of pluripotent stem cells were clearly established by the finding that embryonic stem (ES) cells lacking proteins required for miRNA biogenesis exhibit defects in proliferation and differentiation. Subsequently, the function of numerous miRNAs has been shown to control the fate of ES cells and to directly influence critical gene regulatory networks controlled by pluripotency factors Sox2, Oct4, and Nanog. Moreover, a growing list of tissue-specific miRNAs, which are silenced or not processed fully in ES cells, has been found to promote differentiation upon their expression and proper processing. The importance of miRNAs for ES cells is further indicated by the exciting discovery that specific miRNA mimics or miRNA inhibitors promote the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Although some progress has been made during the past two years in our understanding of the contribution of specific miRNAs during reprogramming, further progress is needed since it is highly likely that miRNAs play even wider roles in the generation of iPS cells than currently appreciated. This review examines recent developments related to the roles of miRNAs in the biology of pluripotent stem cells. In addition, we posit that more than a dozen additional miRNAs are excellent candidates for influencing the generation of iPS cells as well as for providing new insights into the process of reprogramming.