Characteristics Of Pluripotent Stem Cells Research Articles

Generation of integration-free human induced pluripotent stem cell line MURAi003-A derived from the peripheral blood mononuclear cells of a donor with homozygous Class I and Class II HLAs (A*11:01, B*46:01; C*01:02; DRB1*09:01; DQB1*03:03)

The human leukocyte antigen (HLA) system comprises cell-surface proteins responsible for the presentation of peptide antigens. HLAs play an essential role in the regulation of the human immune system, and their studies have been crucial to its understanding. To create a sustainable model for the investigation of HLAs, we successfully generated the human iPSC line MURAi003-A derived from the peripheral blood mononuclear cells of a donor with homozygous Class I and Class II HLAs (A*11:01, B*46:01; C*01:02; DRB1*09:01; DQB1*03:03) using non-integrative reprogramming episomes. MURAi003-A exhibited pluripotent stem cell characteristics with consistent demonstrations of pluripotency and expression of stem cell-associated markers.

Establishment of Vietnamese human induced pluripotent stem cell-derived Orexin-A neurons

Narcolepsy type 1 is a neurological disorder that severely affects sleep control due to a decrease in orexin neurons. Although research on neurological diseases, including narcolepsy, is vital for advancing science and improving human life, progress is hindered by the challenge of obtaining human brain samples as well as the difficulty in culturing neuronal cell lines. To overcome this obstacle, we generated Orexin-A neurons from human induced pluripotent stem cells (hiPSCs) by utilising a differentiation medium supplemented with N-acetyl-D-mannosamine (ManNAc) and Bone morphogenetic protein 4 (BMP4). The cells obtained from our study were characterised using immunofluorescence staining, real-time PCR, and competitive ELISA technique. The results show that our research successfully generated a hiPSC line with pluripotent stem cell characteristics. Furthermore, Orexin-A neurons derived from hiPSCs showed positive results for NCAM1 and TUBB3 markers, an increase in HCRT gene expression, and the capacity to secrete Orexin-A. These promising findings suggest that our hiPSC-derived Orexin-A neurons could serve as a foundation for cell-based disease modelling and further investigations into the underlying mechanisms of related neurological disorders.

Hypoimmunogenic human iPSCs expressing HLA-G, PD-L1, and PD-L2 evade innate and adaptive immunity

BackgroundThe human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body, theoretically. Therefore, hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study, we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch.MethodsFirst, we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then, a HyPSC clone was generated by introducing exogenous β-2-microglobulin (B2M), HLA-G, PD-L1, and PD-L2 genes, and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed.ResultsWe found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover, the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs), functional endothelial cells, and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further, we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo.ConclusionThe HLA class Ia/II DKO hiPSCs armed with HLA-G, PD-L1, PD-L2, and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.

HISTOLOGICAL STRUCTURE AND FUNCTIONAL PROPERTIES OF THE TUNICA ALBUGINEA OF THE OVARY

Just below the surface epithelium of the ovary is the tunica albuginea, which is a tight irregular connective tissue structure that gives the tissue its white color and contains fibroblast cells. Tunica albuginea, which is more resistant to environmental factors, contains fewer cells and is rich in collagen fibers, is observed as the niche of preantral follicles. It has been observed that fibroblasts forming collagen fibers provide the development of follicles with the paracrine factors and cytokines they secrete and function as a nourishing cell layer. After the graaf follicle forms the corpus luteum, fibroblasts in the adjacent tunica albuginea proliferate and the thickness of this structure increases. The tunica albuginea undergoes frequent renewal due to the corpus luteum structures formed in rats. When the corpus luteum is formed, new capillaries and venules are formed in the adjacent tunica albuginea. This structure may be a suitable model for investigating the migration of cells from the bone marrow to the ovary via vessels. It can be suggested that mesenchymal cells and very small embryonic/epiblast-like stem cells (VSELs), which show pluripotent stem cell characteristics, may migrate from the bone marrow to the tunica albuginea through vascular structures and that the bone marrow may be the source of these cells, which have been previously shown to be present in the ovary. The aim of this review is to examine the effects of the tunica albuginea on the development of follicles and the dynamic structure of the ovary.

Using Liver Organoids as Models to Study the Pathobiology of Rare Liver Diseases.

Liver organoids take advantage of several important features of pluripotent stem cells that self-assemble in a three-dimensional culture matrix and reproduce many aspects of the complex organization found within their native tissue or organ counterparts. Compared to other 2D or 3D in vitro models, organoids are widely believed to be genetically stable or docile structures that can be programmed to virtually recapitulate certain biological, physiological, or pathophysiological features of original tissues or organs in vitro. Therefore, organoids can be exploited as effective substitutes or miniaturized models for the study of the developmental mechanisms of rare liver diseases, drug discovery, the accurate evaluation of personalized drug responses, and regenerative medicine applications. However, the bioengineering of organoids currently faces many groundbreaking challenges, including a need for a reasonable tissue size, structured organization, vascularization, functional maturity, and reproducibility. In this review, we outlined basic methodologies and supplements to establish organoids and summarized recent technological advances for experimental liver biology. Finally, we discussed the therapeutic applications and current limitations.

Generation of an iPSC line (FINi001-A) from a girl with developmental and epileptic encephalopathy due to a heterozygous gain-of-function p.R1882Q variant in the voltage-gated sodium channel Nav1.2 protein encoded by the SCN2A gene

A range of epilepsies, including the most severe group of developmental and epileptic encephalopathies (DEEs), are caused by gain-of-function variants in voltage-gated channels. Here we report the generation and characterisation of an iPSC cell line from the fibroblasts of a girl with early infantile DEE carrying heterozygous missense gain-of-function mutation (R1882Q) in Nav1.2(SCN2A) protein, using transient transfection with a single mRNA molecule. The established iPSC line displays typical human primed pluripotent stem cell characteristics: typical colony morphology and robust expression of pluripotency-associated marker genes, ability to give rise to derivatives of all three embryonic germ layers, and normal karyotype without any SNP array-detectable copy number variations. We anticipate that this iPSC line will be useful for the development of neuronal hyperactivity-caused human stem cell-based DEE models, advancing both understanding and potential therapy development for this debilitating condition.

Derivation of MUSIi016-A iPSCs from peripheral blood with blood type O Rh positive

MUSIi016-A, a human induced pluripotent stem cell (iPSC), generated from peripheral blood mononuclear cells of a healthy blood group O Rh positive donor was reprogrammed using Sendai viral vectors containing Yamanaka’s factors. MUSIi016-A iPSC showed pluripotent stem cell characteristics, highly expressed pluripotent markers, and a capacity to differentiate into all three embryonic cell lineages. This iPSC can be used as a model for the generation of blood cells in vitro.

CRISPR/Cas9 mediated approach to generate YAP-depleted human embryonic stem cell line (MUSIe002-A-1)

Yes-associated protein (YAP), an important effector protein of the Hippo signaling pathway, acts as a molecular switch in controlling cell proliferation and apoptosis. In this study, a YAP-targeted isogenic sub-clone of the MUSIe002-A was generated, designated as MUSIe002-A-1. The MUSIe002-1 cell line had normal pluripotent stem cell characteristics and karyotype. Its ability to differentiate into three germ layers was confirmed. As reduction of YAP does not disturb the pluripotency of hESCs, this cell line serves as a valuable model to extrapolate the functional role of YAP in stem cell biology and its applications.

Locus-Specific Isolation of the Nanog Chromatin Identifies Regulators Relevant to Pluripotency of Mouse Embryonic Stem Cells and Reprogramming of Somatic Cells

Pluripotency is a crucial feature of pluripotent stem cells, which are regulated by the core pluripotency network consisting of key transcription factors and signaling molecules. However, relatively less is known about the molecular mechanisms that modify the core pluripotency network. Here we used the CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) to unbiasedly isolate proteins assembled on the Nanog promoter in mouse embryonic stem cells (mESCs), and then tested their functional relevance to the maintenance of mESCs and reprogramming of somatic cells. Gene ontology analysis revealed that the identified proteins, including many RNA-binding proteins (RBPs), are enriched in RNA-related functions and gene expression. ChIP-qPCR experiments confirmed that BCLAF1, FUBP1, MSH6, PARK7, PSIP1, and THRAP3 occupy the Nanog promoter region in mESCs. Knockdown experiments of these factors show that they play varying roles in self-renewal, pluripotency gene expression, and differentiation of mESCs as well as in the reprogramming of somatic cells. Our results show the utility of unbiased identification of chromatin-associated proteins on a pluripotency gene in mESCs and reveal the functional relevance of RBPs in ESC differentiation and somatic cell reprogramming.

Isolation of Swine Bone Marrow Lin-/CD45-/CD133 + Cells and Cardio-protective Effects of its Exosomes

BackgroundThe identification in murine bone marrow (BM) of CD133 + /Lin-/CD45- cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the swine BM. Heart failure is the terminal stage of many cardiovascular diseases, and its key pathological basis is cardiac fibrosis (CF). Research showed that stem cell derived exosomes may play a critical role in cardiac fibrosis. The effect of exosomes (Exos) on CF has remained unclear.ObjectiveTo establish an isolation and amplification method of CD133 + /Lin-/CD45- cells from newbron swine BM in vitro, explore an highly efficient method to enrich swine bone marrow derived CD133 + /Lin-/CD45- cells and probe into their biological characteristics further. Furher more, to extract exosomes from it and explore its effect on CF.MethodsThe mononuclear cells isolated from swine bone marrow by red blood cell (RBC) lysing buffer were coated by adding FcR blocking solution and coupled with CD133 antibody immunomagnetic beads, obtaining CD133 + cell group via Magnetic Activated Cell Sorting (MACS). In steps, the CD133 + /Lin-/CD45- cells were collected by fluorescence-activated cell sorting (FACS) labeled with CD133, Lin and CD45 antibodies, which were cultured and amplified in vitro. The biological features of CD133 + /Lin-/CD45- cells were studied in different aspects, including morphological trait observed with inverted microscope, ultrastructural characteristics observed under transmission electron microscope, expression of pluripotent markersidentified by immunofluorescent staining and Alkaline phosphatase staining.The Exos were extracted using a sequential centrifugation approach and its effects on CF were analyzed in Angiotensin II (Ang-II) induced-cardiac fibrosis in vivo. Rats in each group were treated for 4 weeks, and 2D echocardiography was adopted to evaluate the heart function. The degree of cardiac fibrosis was assessed by Hematoxylin–Eosin (HE) and Masson's trichrome staining.ResultsThe CD133 + /Lin-/CD45- cells accounted for about 0.2%-0.5% of the total mononuclear cells isolated from swine bone marrow. The combination of MACS and FACS to extract CD133 + /Lin-/CD45- cells could improved efficiency and reduced cell apoptosis. The CD133 + /Lin-/CD45- cells featured typical traits of pluripotent stem cells, the nucleus is large, mainly composed of euchromatin, with less cytoplasm and larger nucleoplasmic ratio, which expressed pluripotent markers (SSEA-1, Oct-4, Nanog and Sox-2) and alkaline phosphatase staining was positive.Animal experiment indicated that the cardiac injury related indexes (BNP、cTnI、CK-MB and TNF-α), the expression of key gene Smad3 and the degree of cardiac fibrosis in Exo treatment group were significantly reduced compared with the control group. 4 weeks after the treatment, cardiac ejection fraction (EF) value in the model group showed a remarkable decrease, indicating the induction of HF model. While Exo elevated the EF values, demonstrating cardio-protective effects.ConclusionThe CD133 + /Lin-/CD45- cells derived from swine bone marrow were successfully isolated and amplified, laying a good foundation for further research on this promising therapeutic cell. The Exos may be a promising potential treatment strategy for CF.Graphical

Generation of human induced pluripotent stem cell line from peripheral blood mononuclear cells from an activated phosphoinositide 3-kinase δ syndrome patient

Activated phosphoinositide 3-kinase δ syndrome (APDS) is a rare autosomal dominant primary immunodeficiency disease (PID) which was caused by the acquired mutation of PIK3CD gene. In this study, we generated a human induced pluripotent stem cell (hiPSC) line CHCMUi001-A from the peripheral blood mononuclear cells (PBMCs) of a APDS patient, who has a heterozygous mutation (c.3061 G > A) in the PIK3CD gene. This iPSC line presented a normal karyotype and exhibited characteristics of pluripotent stem cells. This iPSC line can be very useful for not only studying disease mechanisms but also developing new potential clinical treatments for APDS patients.

Generation of an iPSC line (SMCPGi001-A) from a patient with Bain type X-linked mental retardation syndrome carrying HNRNPH2 gene mutation

Bain type X-linked mental retardation syndrome is an X-linked dominant neurodevelopmental disorder characterized by psychomotor developmental delay and intellectual disability. The rare syndrome is caused by HNRNPH2 gene mutation. In this study, the iPSC cell line (SMCPGi001-A) was acquired by Sendai virus-mediated iPSC reprogramming from the peripheral blood mononuclear cells (PBMCs) obtained from a 1-year-old girl with de novo p.R206W mutation in the HNRNPH2 gene. The identification experiments of stemness and differentiation potential of three germ layers showed that the cell line had pluripotent stem cell characteristics and the potential of tridermal differentiation.

Induced pluripotent stem cells from subjects with Lesch-Nyhan disease

Lesch-Nyhan disease (LND) is an inherited disorder caused by pathogenic variants in the HPRT1 gene, which encodes the purine recycling enzyme hypoxanthine–guanine phosphoribosyltransferase (HGprt). We generated 6 induced pluripotent stem cell (iPSC) lines from 3 individuals with LND, along with 6 control lines from 3 normal individuals. All 12 lines had the characteristics of pluripotent stem cells, as assessed by immunostaining for pluripotency markers, expression of pluripotency genes, and differentiation into the 3 primary germ cell layers. Gene expression profiling with RNAseq demonstrated significant heterogeneity among the lines. Despite this heterogeneity, several anticipated abnormalities were readily detectable across all LND lines, including reduced HPRT1 mRNA. Several unexpected abnormalities were also consistently detectable across the LND lines, including decreases in FAR2P1 and increases in RNF39. Shotgun proteomics also demonstrated several expected abnormalities in the LND lines, such as absence of HGprt protein. The proteomics study also revealed several unexpected abnormalities across the LND lines, including increases in GNAO1 decreases in NSE4A. There was a good but partial correlation between abnormalities revealed by the RNAseq and proteomics methods. Finally, functional studies demonstrated LND lines had no HGprt enzyme activity and resistance to the toxic pro-drug 6-thioguanine. Intracellular purines in the LND lines were normal, but they did not recycle hypoxanthine. These cells provide a novel resource to reveal insights into the relevance of heterogeneity among iPSC lines and applications for modeling LND.

Establishment of a novel human iPSC line (SDQLCHi032-A) derived from a patient with primary hypertrophic osteoarthropathy caused by HPGD homozygous mutation.

Primary hypertrophic osteoarthropathy, autosomal recessive type 1 (PHOAR1, MIM259100) is caused by mutations in the 15-hydroxyprostaglandin dehydrogenase gene (HPGD, MIM601688) on chromosome 4q34. An induced pluripotent stem cells (iPSCs) line was generated in our lab from peripheral blood mononuclear cells (PBMCs) of a 2-year-7-month-old girl with PHOAR1 carrying a homozygous mutation of c.310_311del in HPGD. The expression of pluripotency markers, absence of episomal vectors, preservation of normal karyotype, the potential of trilineage differentiation in vitro, were confirmed in the obtained iPSCs line.

Establishment of iPSC (KRIBBi001-A) from CD34+ group O D-negative bone marrow blood.

Human induced pluripotent stem cells with indefinite propagation in vitro provide a potential donor source of cells including erythroid cells for human therapy. Since group O D-negative (RhD-) blood cells are considered as universal donors for transfusion, it is compelling to derive iPSC line from group O/RhD- sample as a new cellular source to generate universal RBCs. The resulting iPSC line derived from group O/RhD- somatic source showed typical features of pluripotent stem cells and could provide an unprecedented cellular tool to develop universal therapeutics for blood transfusion.

Generation of the human induced pluripotent stem cell line (SHAMUi001-A) carrying the heterozygous c.-128G>T mutation in the 5'-UTR of the ANKRD26 gene.

Thrombocytopenia 2 (THC2) is a major type of inherited thrombocytopenia caused by the persistent ANKRD26 expression during the late stage of megakaryocytopoiesis. For the first time, we generated a human induced pluripotent stem cell (hiPSC) line SHAMUi001-A from the bone marrow hematopoietic progenitor cells of a THC2 patient, who has a heterozygous mutation (c.-128G>T) in the 5'-UTR of ANKRD26 gene. SHAMUi001-A cells retain the mutation, display pluripotent stem cell characteristics, and have a normal female karyotype. This disease-specific hiPSC line will be a useful model for THC2 research.

Activation of Wnt/β-Catenin Signaling Pathway Enhances the Derivation of Buffalo (Bubalus bubalis) Embryonic Stem Cell-Like Cells.

Wnt/β-Catenin signaling pathway plays an important role in maintaining self-renewal and pluripotency of human and mouse embryonic stem cells (ESCs). Activation of Wnt/β-Catenin signaling pathway by glycogen synthase kinase-3 (GSK3) inhibitor, the Wnt signaling agonist, could maintain the pluripotency of human and mouse ESCs in the presence of serum. However, the role of signaling pathway in the derivation of buffalo ESCs remains unclear. In this study, we used GSK3 inhibitors (6-bromoindirubin-3'-oxime [BIO] and CHIR99021) and investigated the effect of Wnt/β-Catenin activation on colony formation, proliferation, self-renewal, and pluripotency of Chinese swamp buffalo (buffalo) embryonic stem cell-like cells (ES-like cells), which were isolated from blastocysts. The results showed that buffalo ES-like cells displayed typical morphological characteristics of pluripotent stem cells: positive for alkaline phosphatase staining, expression of pluripotent markers, including OCT4, SOX2, SSEA-1, SSEA-4, LIN28, CH1, NANOG, and the proliferative markers, PCNA and C-MYC. Furthermore, activation of Wnt/β-Catenin signaling pathway by GSK3 inhibitors could promote colony formation and proliferation of buffalo ES-like cells and maintain their undifferentiated state, and upregulate the expression levels of pluripotent-related genes and proliferation-related genes. These results indicated that Wnt/β-Catenin signaling pathway plays an important role in the derivation and pluripotency of buffalo ES-like cells.

Partially Reprogrammed Induced Pluripotent Stem Cells Using MicroRNA Cluster miR-302s in Guangxi Bama Minipig Fibroblasts.

Pig-induced pluripotent stem cells (piPSCs) have great potential application in regenerative medicine. The miR-302s cluster alone has been shown to reprogram mouse and human somatic cells into induced pluripotent stem cells (iPSCs) without exogenous transcription factors. However, miR-302s alone have not been reported to reprogram cells in large livestock. In this study, we induced pig somatic cells into partially reprogrammed piPSCs using overexpression of the miR-302s cluster (miR-302s-piPSC) and investigated the early reprogramming events during the miRNA induction process. The results showed that miR-302s-piPSCs exhibited some characteristics of pluripotent stem cells including expression of pluripotency markers-particularly, efficient activation of endogenous OCT4-and differentiation to the three germ layers in vitro. During the early reprogramming process, somatic cells first underwent epithelial-mesenchymal transition and then mesenchymal-epithelial transition to eventually form miR-302s-piPSCs. These data show, for the first time, that single factor miR-302s successfully induced pig somatic cells into miR-302s-piPSCs. This study provides a new tool and research direction for the induction of pluripotent stem cells in a large livestock.

Glutamine independence is a selectable feature of pluripotent stem cells.

Most rapidly proliferating mammalian cells rely on the oxidation of exogenous glutamine to support cell proliferation. We previously found that culture of mouse embryonic stem cells (ESCs) in the presence of inhibitors against MEK and GSK3β to maintain pluripotency reduces cellular reliance on glutamine for tricarboxylic acid (TCA) cycle anaplerosis, enabling ESCs to proliferate in the absence of exogenous glutamine. Here we show that reduced dependence on exogenous glutamine is a generalizable feature of pluripotent stem cells. Enhancing self-renewal, through either overexpression of pluripotency-associated transcription factors or altered signal transduction, decreases the utilization of glutamine-derived carbons in the TCA cycle. As a result, cells with the highest potential for self-renewal can be enriched by transient culture in glutamine-deficient media. During pluripotent cell culture or reprogramming to pluripotency, transient glutamine withdrawal selectively leads to the elimination of non-pluripotent cells. These data reveal that reduced dependence on glutamine anaplerosis is an inherent feature of self-renewing pluripotent stem cells and reveal a simple, non-invasive mechanism to select for mouse and human pluripotent stem cells within a heterogeneous population during both ESC passage and induced pluripotent cell reprogramming.

Expression of mutant mRNA and protein in pancreatic cells derived from MODY3- iPS cells.

Maturity-onset diabetes of the young (MODY) is a heterozygous monogenic diabetes; more than 14 disease genes have been identified. However, the pathogenesis of MODY is not fully understood because the patients’ pancreatic beta cells are inaccessible. To elucidate the pathology of MODY, we established MODY3 patient-derived iPS (MODY3-iPS) cells using non-integrating Sendai virus (SeV) vector and examined the mutant mRNA and protein of HNF1A (Hepatocyte Nuclear factor 1A) after pancreatic lineage differentiation. Our patient had a cytosine insertion in the HNF1A gene (P291fsinsC) causing frameshift and making a premature termination codon (PTC). We confirmed these MODY3-iPS cells possessed the characteristics of pluripotent stem cells. After we differentiated them into pancreatic beta cells, transcripts of HNF1A gene were cloned and sequenced. We found that P291fsinsC mutant transcripts were much less frequent than wild ones, but they increased after adding cycloheximide (CHX) to the medium. These results suggested that mutant mRNA was destroyed by nonsense-mediated mRNA decay (NMD). Moreover, we were not able to detect any band of mutant proteins in pancreatic lineage cells which were differentiated from MODY3-iPSCs by western blot (WB) analysis. A scarcity of the truncated form of mutant protein may indicate that MODY3 might be caused by a haplo-insufficiency effect rather than a dominant negative manner.