Transcriptomic landscape of human induced pluripotent stem cell-derived osteogenic differentiation identifies a regulatory role of KLF16.

Osteogenic differentiation is essential for bone development and metabolism, but the underlying gene regulatory networks have not been well investigated. We differentiated mesenchymal stem cells, derived from 20 human induced pluripotent stem cell lines, into preosteoblasts and osteoblasts, and performed systematic RNA-seq analyses of 60 samples for differential gene expression. We noted a highly significant correlation in expression patterns and genomic proximity among transcription factor (TF) and long noncoding RNA (lncRNA) genes. We identified TF-TF regulatory networks, regulatory roles of lncRNAs on their neighboring coding genes for TFs and splicing factors, and differential splicing of TF, lncRNA, and splicing factor genes. TF-TF regulatory and gene co-expression network analyses suggested an inhibitory role of TF KLF16 in osteogenic differentiation. We demonstrate that in vitro overexpression of human KLF16 inhibits osteogenic differentiation and mineralization, and in vivo Klf16+/- mice exhibit increased bone mineral density, trabecular number, and cortical bone area. Thus, our model system highlights the regulatory complexity of osteogenic differentiation and identifies novel osteogenic genes.

Reprogramming of human peripheral blood mononuclear cells into induced mesenchymal stromal cells using non-integrating vectors.

Mesenchymal stromal cells (MSCs) have great value in cell therapies. The MSC therapies have many challenges due to its inconsistent potency and limited quantity. Here, we report a strategy to generate induced MSCs (iMSCs) by directly reprogramming human peripheral blood mononuclear cells (PBMCs) with OCT4, SOX9, MYC, KLF4, and BCL-XL using a nonintegrating episomal vector system. While OCT4 was not required to reprogram PBMCs into iMSCs, omission of OCT4 significantly impaired iMSC functionality. The omission of OCT4 resulted in significantly downregulating MSC lineage specific and mesoderm-regulating genes, including SRPX, COL5A1, SOX4, SALL4, TWIST1. When reprogramming PBMCs in the absence of OCT4, 67 genes were significantly hypermethylated with reduced transcriptional expression. These data indicate that transient expression of OCT4 may serve as a universal reprogramming factor by increasing chromatin accessibility and promoting demethylation. Our findings represent an approach to produce functional MSCs, and aid in identifying putative function associated MSC markers.

Predicting individual-specific cardiotoxicity responses induced by tyrosine kinase inhibitors.

Introduction: Tyrosine kinase inhibitor drugs (TKIs) are highly effective cancer drugs, yet many TKIs are associated with various forms of cardiotoxicity. The mechanisms underlying these drug-induced adverse events remain poorly understood. We studied mechanisms of TKI-induced cardiotoxicity by integrating several complementary approaches, including comprehensive transcriptomics, mechanistic mathematical modeling, and physiological assays in cultured human cardiac myocytes. Methods: Induced pluripotent stem cells (iPSCs) from two healthy donors were differentiated into cardiac myocytes (iPSC-CMs), and cells were treated with a panel of 26 FDA-approved TKIs. Drug-induced changes in gene expression were quantified using mRNA-seq, changes in gene expression were integrated into a mechanistic mathematical model of electrophysiology and contraction, and simulation results were used to predict physiological outcomes. Results: Experimental recordings of action potentials, intracellular calcium, and contraction in iPSC-CMs demonstrated that modeling predictions were accurate, with 81% of modeling predictions across the two cell lines confirmed experimentally. Surprisingly, simulations of how TKI-treated iPSC-CMs would respond to an additional arrhythmogenic insult, namely, hypokalemia, predicted dramatic differences between cell lines in how drugs affected arrhythmia susceptibility, and these predictions were confirmed experimentally. Computational analysis revealed that differences between cell lines in the upregulation or downregulation of particular ion channels could explain how TKI-treated cells responded differently to hypokalemia. Discussion: Overall, the study identifies transcriptional mechanisms underlying cardiotoxicity caused by TKIs, and illustrates a novel approach for integrating transcriptomics with mechanistic mathematical models to generate experimentally testable, individual-specific predictions of adverse event risk.

Ex vivo reprogramming of human hematopoietic stem cells is accompanied by increased transcripts of genes regulating metabolic integrity.

The regenerative potential of human hematopoietic stem cells (HSCs) is functionally defined by their ability to provide life-long blood cell production and to repopulate myeloablated allogeneic transplant recipients. The expansion of HSC numbers is dependent not only on HSC divisions but also on a coordinated adaptation of HSCs to metabolic stress. These variables are especially critical during the ex vivo culture of HSCs with cytokine combinations, which frequently results in HSC exhaustion. We have previously reported that human CD34+ hematopoietic stem and progenitor cells (HSPCs) can be efficiently reprogrammed ex vivo and that the number of phenotypic HSCs with long-term repopulation capacity is expanded in the presence of a combination of cytokines and an epigenetic modifier. Here, we present evidence that ex vivo HSC reprogramming and maintenance is accompanied by increased transcripts of genes regulating metabolic integrity, including SIRT1 and SIRT3.

Functional Effects of Cardiomyocyte Injury in COVID-19.

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System show that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrated cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of interleukins (ILs) with clinical findings related to laboratory values in COVID-19 patients to identify plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes from healthy human subjects with SARS-CoV-2 in the absence and presence of IL-6 and IL-1ß. Infection resulted in increased numbers of multinucleated cells. Interleukin treatment and infection resulted in disorganization of myofibrils, extracellular release of troponin I, and reduced and erratic beating. Infection resulted in decreased expression of mRNA encoding key proteins of the cardiomyocyte contractile apparatus. Although interleukins did not increase the extent of infection, they increased the contractile dysfunction associated with viral infection of cardiomyocytes, resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health System show that a significant portion of COVID-19 patients without history of heart disease have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection might underlie heart disease in COVID-19 patients. IMPORTANCE SARS-CoV-2 infects multiple organs, including the heart. Analyses of hospitalized patients show that a substantial number without prior indication of heart disease or comorbidities show significant injury to heart tissue, assessed by increased levels of troponin in blood. We studied the cell biological and physiological effects of virus infection of healthy human iPSC-derived cardiomyocytes in culture. Virus infection with interleukins disorganizes myofibrils, increases cell size and the numbers of multinucleated cells, and suppresses the expression of proteins of the contractile apparatus. Viral infection of cardiomyocytes in culture triggers release of troponin similar to elevation in levels of COVID-19 patients with heart disease. Viral infection in the presence of interleukins slows down and desynchronizes the beating of cardiomyocytes in culture. The cell-level physiological changes are similar to decreases in left ventricular ejection seen in imaging of patients' hearts. These observations suggest that direct injury to heart tissue by virus can be one underlying cause of heart disease in COVID-19.

A library of induced pluripotent stem cells from clinically well-characterized, diverse healthy human individuals.

A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a useful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically healthy human individuals who range in age from 22 to 61 years. The hiPSCs match the karyotype and short tandem repeat identities of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole-genome sequencing data for one hiPSC clone from each individual, genomic ancestry determination, and analysis of mendelian disease genes and risks. We document similar transcriptomic profiles, single-cell RNA-sequencing-derived cell clusters, and physiology of cardiomyocytes differentiated from multiple independent hiPSC lines. This extensive characterization makes this hiPSC library a valuable resource for many studies on human biology.

Evaluation of a clinical-grade, cryopreserved, ex vivo-expanded stem cell product from cryopreserved primary umbilical cord blood demonstrates multilineage hematopoietic engraftment in mouse xenografts.

BACKGROUND AIMS: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for a wide range of malignant and genetic disorders of the hematopoietic and immune systems. Umbilical cord blood (UCB) is a readily available source of stem cells for allo-HSCT, but the small fixed number of hematopoietic stem and progenitor cells (HSPCs) found in a single unit limits its widespread use in adult recipients. The authors have previously reported that culturing UCB-CD34+ cells in serum-free media supplemented with a combination of cytokines and the histone deacetylase inhibitor valproic acid (VPA) led to expansion of the numbers of functional HSPCs. Such fresh expanded product has been advanced to the clinic and is currently evaluated in an ongoing clinical trial in patients with hematological malignancies undergoing allo-HSCT. Here the authors report on the cryopreservation of this cellular product under current Good Manufacturing Practice (cGMP). METHODS: cGMP VPA-mediated expansion was initiated with CD34+ cells isolated from cryopreserved primary UCB collections, and the functionality after a second cryopreservation step of the expanded product evaluted in vitro and in mouse xenografts. RESULTS: The authors found that the cryopreserved VPA-expanded grafts were characterized by a high degree of viability, retention of HSPC phenotypic subtypes and maintenance of long-term multilineage repopulation capacity in immunocompromised mice. All cellular and functional parameters tested were comparable between the fresh and cryopreserved VPA-expanded cellular products. CONCLUSIONS: The authors' results demonstrate and support the practicality of cryopreservation of VPA-expanded stem cell grafts derived from UCB-CD34+ cells for clinical utilization.

Ex Vivo Expansion of Adult Hematopoietic Stem and Progenitor Cells with Valproic Acid.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic stem cell transplantation but lack a matched donor. However, the limited number of HSCs within each UCB unit remains a major challenge for their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of human HSCs in ex vivo cultures initiated with CD34+ cells isolated from UCBs can overcome this limitation. The method described here utilizes a deacetylase inhibitor, valproic acid (VPA), to rapidly expand to a high degree the numbers of functional HSCs and committed progenitors (HPCs). The expanded HSCs are capable of establishing both short-term and long-term multilineage hematopoietic reconstitution. This highly reproducible and simple protocol can be also applied to expansion of both HSCs and HPCs from different sources including the bone marrow and peripheral blood.

Physiology of cardiomyocyte injury in COVID-19.

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System shows that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrate cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with SARS-CoV-2 in the presence of interleukins, with clinical findings, to investigate plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes, from healthy human subjects, with SARS-CoV-2 in the absence and presence of interleukins. We find that interleukin treatment and infection results in disorganization of myofibrils, extracellular release of troponin-I, and reduced and erratic beating. Although interleukins do not increase the extent, they increase the severity of viral infection of cardiomyocytes resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health system show that a significant portion of COVID-19 patients without prior history of heart disease, have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection can underlie the heart disease in COVID-19 patients.

A human H1-HBB11-GFP reporter embryonic stem cell line (WAe001-A-2) generated using TALEN-based genome editing.

Hemoglobin production during mammalian development is characterized by temporal switches of the genes coding for the α- and ß-globin chains. Defects in this controlled process can lead to hemoglobinapathies such as sickle cell disease and ß-thalassemia. The ability of human embryonic stem cells (hESC) to proceed through hematopoiesis could provide a clinically useful source of red blood cells. However, hESC-derived red cells exhibit an embryonic/fetal, but not adult, mode of hemoglobin expression. The resource described here is a hESC line engineered to express a reporter from its adult globin promoter, providing a screening platform for small molecules that lead to efficient induction of adult globin.

Induction of developmental hematopoiesis mediated by transcription factors and the hematopoietic microenvironment.

The induction of hematopoiesis in various cell types via transcription factor (TF) reprogramming has been demonstrated by several strategies. The eventual goal of these approaches is to generate a product for unmet needs in hematopoietic cell transplantation therapies. The most successful strategies hew closely to clues provided from developmental hematopoiesis in terms of factor expression and environmental cues. In this review, we aim to summarize the TFs that play important roles in developmental hematopoiesis primarily and to also touch on adult hematopoiesis. Several aspects of cellular and molecular biology coalesce in this process, with TFs and surrounding cellular signals playing a major role in the overall development of the hematopoietic lineage. We attempt to put these elements into the context of reprogramming and highlight their roles.

Limited Mitochondrial Activity Coupled With Strong Expression of CD34, CD90 and EPCR Determines the Functional Fitness of ex vivo Expanded Human Hematopoietic Stem Cells.

Ex vivo expansion strategies of human hematopoietic stem cell (HSC) grafts with suboptimal stem cell dose have emerged as promising strategies for improving outcomes of HSC transplantation in patients with hematological malignancies. While exposure of HSCs to ex vivo cultures expands the number of phenotypically identifiable HSCs, it frequently alters the transcriptomic and metabolic profiles, therefore, compromising their long-term (LT) hematopoietic reconstitution capacity. Within the heterogeneous pool of expanded HSCs, the precise phenotypic, transcriptomic and metabolic profile and thus, the identity of HSCs that confer LT repopulation potential remains poorly described. Utilizing valproic acid (VPA) in ex vivo cultures of umbilical cord blood (UCB)-CD34+ cells, we demonstrate that expanded HSCs phenotypically marked by expression of the stem cell markers CD34, CD90 and EPCR (CD201) are highly enriched for LT-HSCs. Furthermore, we report that low mitochondrial membrane potential, and, hence, mitochondrial activity distinguishes LT-HSCs within the expanded pool of phenotypically defined HSCs. Remarkably, such reduced mitochondrial activity is restricted to cells with the highest expression levels of CD34, CD90 and EPCR phenotypic markers. Together, our findings reveal that high expression of CD34, CD90 and EPCR in conjunction with low mitochondrial activity is critical for identification of functional LT-HSCs generated within ex vivo expansion cultures.

Genomic Integrity Safeguards Self-Renewal in Embryonic Stem Cells.

A multitude of signals are coordinated to maintain self-renewal in embryonic stem cells (ESCs). To unravel the essential internal and external signals required for sustaining the ESC state, we expand upon a set of ESC pluripotency-associated phosphoregulators (PRs) identified previously by short hairpin RNA (shRNA) screening. In addition to the previously described Aurka, we identify 4 additional PRs (Bub1b, Chek1, Ppm1g, and Ppp2r1b) whose depletion compromises self-renewal and leads to consequent differentiation. Global gene expression profiling and computational analyses reveal that knockdown of the 5 PRs leads to DNA damage/genome instability, activating p53 and culminating in ESC differentiation. Similarly, depletion of genome integrity-associated genes involved in DNA replication and checkpoint, mRNA processing, and Charcot-Marie-Tooth disease lead to compromise of ESC self-renewal via an increase in p53 activity. Our studies demonstrate an essential link between genomic integrity and developmental cell fate regulation in ESCs.

Oncogenic role of SFRP2 in p53-mutant osteosarcoma development via autocrine and paracrine mechanism.

Osteosarcoma (OS), the most common primary bone tumor, is highly metastatic with high chemotherapeutic resistance and poor survival rates. Using induced pluripotent stem cells (iPSCs) generated from Li-Fraumeni syndrome (LFS) patients, we investigate an oncogenic role of secreted frizzled-related protein 2 (SFRP2) in p53 mutation-associated OS development. Interestingly, we find that high SFRP2 expression in OS patient samples correlates with poor survival. Systems-level analyses identified that expression of SFRP2 increases during LFS OS development and can induce angiogenesis. Ectopic SFRP2 overexpression in normal osteoblast precursors is sufficient to suppress normal osteoblast differentiation and to promote OS phenotypes through induction of oncogenic molecules such as FOXM1 and CYR61 in a ß-catenin-independent manner. Conversely, inhibition of SFRP2, FOXM1, or CYR61 represses the tumorigenic potential. In summary, these findings demonstrate the oncogenic role of SFRP2 in the development of p53 mutation-associated OS and that inhibition of SFRP2 is a potential therapeutic strategy.

Modeling Hematological Diseases and Cancer With Patient-Specific Induced Pluripotent Stem Cells.

The advent of induced pluripotent stem cells (iPSCs) together with recent advances in genome editing, microphysiological systems, tissue engineering and xenograft models present new opportunities for the investigation of hematological diseases and cancer in a patient-specific context. Here we review the progress in the field and discuss the advantages, limitations, and challenges of iPSC-based malignancy modeling. We will also discuss the use of iPSCs and its derivatives as cellular sources for drug target identification, drug development and evaluation of pharmacological responses.

A Marfan syndrome human induced pluripotent stem cell line with a heterozygous FBN1 c.4082G>A mutation, ISMMSi002-B, for disease modeling.

Fibroblasts of a 28-year-old female with Marfan syndrome (MFS) due to a heterozygous FBN1 c.4082G>A mutation were reprogrammed using the Sendai virus delivery method. The established human induced pluripotent stem cell (hiPSC) line named ISMMSi002-B expresses pluripotency markers, has a normal karyotype, carries the specific FBN1 mutation and is able to differentiate into three germ layers in vitro. ISMMSi002-B has utility in studying MFS pathogenesis, including skeletal abnormalities, cardiomyopathy, and vascular smooth muscle cell dysfunction associated with aortic aneurysm. Furthermore, it can serve as a platform for drug discovery.

A human MIXL1 green fluorescent protein reporter embryonic stem cell line engineered using TALEN-based genome editing.

We have generated a MIXL1-eGFP reporter human embryonic stem cell (hESC) line using TALEN-based genome engineering. This line accurately traces endogenous MIXL1 expression via an eGFP reporter to mesendodermal precursor cells. The utility of the MIXL1-eGFP reporter hESC line lies in the prospective isolation, lineage tracing, and developmental and mechanistic studies of MIXL1+ cell populations.

Genomic imprinting defect in Zfp57 mutant iPS cell lines.

ZFP57 maintains genomic imprinting in mouse embryos and ES cells. To test its roles during iPS reprogramming,we derived iPS clones by utilizing retroviral infection to express reprogramming factors in mouse MEF cells. After analyzing four imprinted regions, we found that parentally derived DNA methylation imprint was largely maintained in the iPS clones with Zfp57 but missing in those without maternal or zygotic Zfp57. Intriguingly, DNA methylation imprint was lost at the Peg1 and Peg3 but retained at the Snrpn and Dlk1-Dio3 imprinted regions in the iPS clones without zygotic Zfp57. This finding will be pursued in future studies.

Hematopoietic Reprogramming In Vitro Informs In Vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells.

Definitive hematopoiesis emerges via an endothelial-to-hematopoietic transition in the embryo and placenta; however, the precursor cells to hemogenic endothelium are not defined phenotypically. We previously demonstrated that the induction of hematopoietic progenitors from fibroblasts progresses through hemogenic precursors that are Prom1(+)Sca1(+)CD34(+)CD45(-) (PS34CD45(-)). Guided by these studies, we analyzed mouse placentas and identified a population with this phenotype. These cells express endothelial markers, are heterogeneous for early hematopoietic markers, and localize to the vascular labyrinth. Remarkably, global gene expression profiles of PS34CD45(-) cells correlate with reprogrammed precursors and establish a hemogenic precursor cell molecular signature. PS34CD45(-) cells are also present in intra-embryonic hemogenic sites. After stromal co-culture, PS34CD45(-) cells give rise to all blood lineages and engraft primary and secondary immunodeficient mice. In summary, we show that reprogramming reveals a phenotype for in vivo precursors to hemogenic endothelium, establishing that direct in vitro conversion informs developmental processes in vivo.

Myeloid Dysregulation in a Human Induced Pluripotent Stem Cell Model of PTPN11-Associated Juvenile Myelomonocytic Leukemia.

Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML). Germline PTPN11 defects cause Noonan syndrome (NS), and specific inherited mutations cause NS/JMML. Here, we report that hematopoietic cells differentiated from human induced pluripotent stem cells (hiPSCs) harboring NS/JMML-causing PTPN11 mutations recapitulated JMML features. hiPSC-derived NS/JMML myeloid cells exhibited increased signaling through STAT5 and upregulation of miR-223 and miR-15a. Similarly, miR-223 and miR-15a were upregulated in 11/19 JMML bone marrow mononuclear cells harboring PTPN11 mutations, but not those without PTPN11 defects. Reducing miR-223's function in NS/JMML hiPSCs normalized myelogenesis. MicroRNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations. Thus, studying an inherited human cancer syndrome with hiPSCs illuminated early oncogenesis prior to the accumulation of secondary genomic alterations, enabling us to discover microRNA dysregulation, establishing a genotype-phenotype association for JMML and providing therapeutic targets.