Changes in Purine Metabolism During Differentiation of Dopamine Neurons from Human Induced Pluripotent Stem Cells.

SummaryInduced pluripotent stem cells (iPSCs) are valuable in disease modeling because of their potential to expand and differentiate into virtually any cell type and recapitulate key aspects of human biology. Functional genomics are genome-wide studies that aim to discover genotype-phenotype relationships, thereby revealing the impact of human genetic diversity on normal and pathophysiology. In this review, we make the case that human iPSCs (hiPSCs) are a powerful tool for functional genomics, since they provide an in vitro platform for the study of population genetics. We describe cutting-edge tools and strategies now available to researchers, including multi-omics technologies, advances in hiPSC culture techniques, and innovations in drug development. Functional genomics approaches based on hiPSCs hold great promise for advancing drug discovery, disease etiology, and the impact of genetic variation on human biology.

To the Editor: The molecular mechanisms and the control of smooth muscle cell (SMC) differentiation have been extensively investigated because of its therapeutic potential.1 To date, different cell types have been used to study SMC differentiation, including a variety of mouse embryonic stem cells,2 adult stem cells,3,4 and others.5 Because several fundamental differences exist between mouse and human embryonic development,6 lack of a good model system to study human SMC differentiation has hampered the progress of translating SMC knowledge to novel clinical therapies. Human embryonic stem (hES) cells provide a valuable source of cells for studying human cell differentiation and developing therapeutic potentials in regenerative medicine. Since the initial report describing the derivation of hES cells,7 a variety of studies have established in vitro differentiation strategies to several lineages. Recently, it has been demonstrated that vascular progenitors derived from hES cells could be differentiated into endothelial cells and SMCs by endothelial …

Chromatin Structure and Gene Expression Programs of Human Embryonic and Induced Pluripotent Stem Cells

Translation: Screening for Novel Therapeutics With Disease-Relevant Cell Types Derived from Human Stem Cell Models

Patient-derived iPSC-cerebral organoid modeling of the 17q11.2 microdeletion syndrome establishes CRLF3 as a critical regulator of neurogenesis.

Lonely death dance of human pluripotent stem cells: ROCKing between metastable cell states

Remodeling Neurodegeneration: Somatic Cell Reprogramming-Based Models of Adult Neurological Disorders

Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.

Engineering of Human Pluripotent Stem Cells by AAV-mediated Gene Targeting

Neurogenesis in the developing neocortex begins with the generation of the preplate, which consists of early-born neurons including Cajal-Retzius (CR) cells and subplate neurons. Here, utilizing the Ebf2-EGFP transgenic mouse in which EGFP initially labels the preplate neurons then persists in CR cells, we reveal the dynamic transcriptome profiles of early neurogenesis and CR cell differentiation. Genome-wide RNA-seq and ChIP-seq analyses at multiple early neurogenic stages have revealed the temporal gene expression dynamics of early neurogenesis and distinct histone modification patterns in early differentiating neurons. We have identified a new set of coding genes and lncRNAs involved in early neuronal differentiation and validated with functional assays in vitro and in vivo. In addition, at E15.5 when Ebf2-EGFP+ cells are mostly CR neurons, single-cell sequencing analysis of purified Ebf2-EGFP+ cells uncovers molecular heterogeneities in CR neurons, but without apparent clustering of cells with distinct regional origins. Along a pseudotemporal trajectory these cells are classified into three different developing states, revealing genetic cascades from early generic neuronal differentiation to late fate specification during the establishment of CR neuron identity and function. Our findings shed light on the molecular mechanisms governing the early differentiation steps during cortical development, especially CR neuron differentiation.

Rapid and Efficient Generation of Functional Motor Neurons From Human Pluripotent Stem Cells Using Gene Delivered Transcription Factor Codes

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.

Human pluripotent stem cells (PSCs) have been utilized as a promising source in regenerative medicine. However, the risk of teratoma formation that comes with residual undifferentiated PSCs in differentiated cell populations is most concerning in the clinical use of PSC derivatives. Here, we report that a monoclonal antibody (mAb) targeting PSCs could distinguish undifferentiated PSCs, with potential teratoma-forming activity, from differentiated PSC progeny. A panel of hybridomas generated from mouse immunization with H9 human embryonic stem cells (hESCs) was screened for ESC-specific binding using flow cytometry. A novel mAb, K312, was selected considering its high stem cell-binding activity, and this mAb could bind to several human induced pluripotent stem cells and PSC lines. Cell-binding activity of K312 was markedly decreased as hESCs were differentiated into embryoid bodies or by retinoic acid treatment. In addition, a cell population negatively isolated from undifferentiated or differentiated H9 hESCs via K312 targeting showed a significantly reduced expression of pluripotency markers, including Oct4 and Nanog. Furthermore, K312-based depletion of pluripotent cells from differentiated PSC progeny completely prevented teratoma formation. Therefore, our findings suggest that K312 is utilizable in improving stem cell transplantation safety by specifically distinguishing residual undifferentiated PSCs.

To explore the role of over-expression of TBX3 and TBX18 in inducing human induced pluripotent stem cells (HiPS) to enrich and differentiate into sinoatrial node-like cells. The expression of stemness markers OCT3/4, SOX2, and NANOG in HiPS was detected by real-time fluorescence quantitative PCR (qRT- PCR), and compared with human embryonic stem cells (hESCs). Immunofluorescence staining was used to observe the expression of HiPS stemness markers OCT3/4, NANOG, SSEA4, and TRA-1-60. The HiPS were directional differentiated into cardiomyocytes, the expressions of ISL1, NK2 homeobox 5 (NKX2-5), ACTN1, and TNNT2 were detected by qRT-PCR, and human adult cardiomyocytes (hACM) were used as positive control. Immunofluorescence staining was used to observe the expressions of NKX2-5, cardiac troponin (cTnT), α-actinin, atria myosin light chain 2A (MLC-2A), and ventricular myosin light chain 2V (MLC-2V). The positive rate of α-actinin was detected by flow cytometry. On the 3rd day after HiPS were differentiated into cardiomyocytes (mesodermal stage), lentiviral over-expressions of sinoatrial node-related genes TBX3 and TBX18 were carried out for 21 days. The relative expressions of specific markers TBX3, TBX18, SHOX2, NKX2-5, HCN4, and HCN1 in sinoatrial node cells were detected by qRT-PCR, and compared with enhanced green fluorescent protein blank virus. OCT3/4, SOX2, and NANOG were highly expressed in HiPS and ESCs, and there was no significant difference in the relative expression of each gene ( P>0.05); OCT3/4 and NANOG were specifically distributed in the nucleus of HiPS, while SSEA4 and TRA-1-60 were distributed in the cell membrane. The relative expressions of ISL1 gene at 5, 7, 21, and 28 days and NKX2-5 gene at 7, 21, and 28 days of HiPS differentiation into cardiomyocytes were significantly higher than those of hACM ( P<0.05), and the relative expressions of ACTN1 and TNNT2 genes at 3, 5, 7, and 21 days of HiPS differentiation into cardiomyocytes were significantly lower than those of hACM ( P<0.05). NKX2-5 was expressed in most of the nuclei, cTnT and α-actinin, MLC-2A and MLC-2V signals were localized in the cytoplasm, presenting a texture-like structure of muscle nodules. Flow cytometry results showed that HiPS was successfully induced to differentiate into cardiomyocytes. The expressions of TBX18, SHOX2, HCN4, and HCN1 in the over-expression TBX3 group were up-regulated when compared with the control group, and difference in the relative expression of SHOX2 gene was significant ( P<0.05); the relative expression of NKX2-5 gene was lower than that in the control group, but there was no significant difference ( P>0.05). There was no significant difference in the relative expression of each gene between the over-expressed TBX18 group and the control group ( P>0.05). HiPS and hESCs have similar pluripotency, and we have established a stable method for maintaining and culturing the stemness of HiPS. A technological platform for the efficient differentiation of HiPS into cardiomyocytes has been successfully established. Although TBX3 and TBX18 do not play a significant role in promoting the enrichment and differentiation of HiPS into sinoatrial node-like cells, TBX3 shows a certain promoting trend, which can be further explored in the future.

PRC1 catalytic unit RING1B regulates early neural differentiation of human pluripotent stem cells