Expression Of Pluripotency-associated Markers Research Articles

Generation of the human induced pluripotent stem cell line (IBKMOLi003-A) from PBMCs of a vascular Ehlers-Danlos syndrome (vEDS) patient carrying the heterozygous nonsense mutation c.430C > T (p.Q105*) in the COL3A1 gene

Ehlers-Danlos syndrome (EDS) belongs to a spectrum of rare heritable connective tissue disorders and is characterised by hyperextensibility, joint hypermobility and tissue fragility. Peripheral blood mononuclear cells (PBMCs) from a vascular EDS (vEDS) patient, known as the rarest EDS subtype, carrying a heterozygous nonsense mutation c.430C > T (p.Q105*) in the COL3A1 gene, which is essential for type III collagen synthesis, were reprogrammed into induced pluripotent stem cells (iPSCs). The generated iPSCs exhibit high expression of pluripotency-associated markers, possess trilineage differentiation capacity and reveal a normal karyotype. This novel patient-specific cell line enables in-depth pathophysiological studies of vEDS.

Generation of human induced pluripotent stem cell lines derived from four patients with a pathogenic ALPK3 variant associated with adult-onset hypertrophic cardiomyopathy (HCM)

Loss of function variants in ALPK3 have been associated with dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). However, the underlying pathomechanism remain largely unknown. Here, we generated human iPSC lines from four HCM patients carrying the heterozygous pathogenic variant in ALPK3 (c.2023delC p.Gln675fs). Peripheral blood mononuclear cells (PBMCs) from patients were reprogrammed to induced pluripotent stem cells (iPSCs) with the Sendai virus-based reprogramming method. All four lines display typical iPSC morphology, normal karyotype, expression of pluripotency-associated markers, and trilineage differentiation potential. These iPSC lines represent a valuable resource of ALPK3 patient-derived iPSC lines to the study ALPK3-associated cardiomyopathy.

Generation of the iPSC line FINi002-A from a male Parkinson's disease patient carrying compound heterozygous mutations in the PRKN gene

The most common cause of autosomal recessive familial Parkinson’s disease (PD) are mutations in the PRKN/PARK2 gene encoding an E3 ubiquitin protein-ligase PARKIN. We report the generation of an iPSC cell line from the fibroblasts of a male PD patient carrying a common missense variant in exon 7 (p.Arg275Trp), and a 133 kb deletion encompassing exon 8, using transiently-present Sendai virus. The established line displays typical human primed iPSC morphology and expression of pluripotency-associated markers, normal karyotype without SNP array-detectable copy number variations and can give rise to derivatives of all three embryonic germ layers. We envisage the usefulness of this iPSC line, carrying a common and well-studied missense mutation in the RING1 domain of the PARKIN protein, for the elucidation of PARKIN-dependent mechanisms of PD using in vitro and in vivo models.

Generation of a ST3GAL3 null mutant induced pluripotent stem cell (iPSC) line (UKWMPi002-A-3) by CRISPR/Cas9 genome editing

Fibroblasts isolated from a skin biopsy of a healthy individual were infected with Sendai virus containing the Yamanaka factors to produce transgene-free human induced pluripotent stem cells (iPSCs). CRISPR/Cas9 was used to generate an isogenic cell line carrying an inactivation of ST3GAL3, a risk gene associated with neurodevelopmental and psychiatric disorders. This ST3GAL3 null mutant (ST3GAL3-/-) iPSC line, which displays the expression of pluripotency-associated markers, the ability to differentiate into cells of the three germ layers in vitro, and a normal karyotype, is a powerful tool to investigate the impact of deficient sialylation of glycoproteins in neural development and plasticity.

Generation of a CRISPR/Cas9-corrected-hiPSC line (DDLABi001-A) from Fabry disease (FD)-derived iPSCs having α-galactosidase (GLA) gene mutation (c.803_806del)

Fabry disease (FD) is a lysosomal storage disorder caused by mutations in GLA gene. Here, GLA mutation (1268fs*1 (c.803_806del)) of FD iPSCs was corrected using the CRISPR-Cas9 gene editing system. The corrected (cor) FD-iPSCs retained normal morphology, karyotype, expression of pluripotency-associated markers, trilineage differentiation potential, and GLA activity. Thus, FD(cor)-iPSCs can be used as valuable tools to study the mechanism how GLA mutation1268fs*1 induces various pathophysiologic phenotypes in FD patients.

Magnetic exposure using Samarium Cobalt (SmCO5) increased proliferation and stemness of human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs)

Despite the extensive reports on the potential hazard of magnetic field (MF) exposures on humans, there are also concurrently reported on the improved proliferative property of stem cells at optimum exposure. However, the effect on mesenchymal stem cells (MSCs) remains unknown. Therefore, we aimed to investigate the impact of induced static MF (SMF) on human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) using Samarium Cobalt (SmCO5). At passage 3, hUC-MSCs (1 × 104) were exposed to 21.6 mT SMF by a direct exposure (DE) showed a significantly higher cell count (p < 0.05) in the growth kinetics assays with the shortest population doubling time relative to indirect exposure and negative control. The DE group was committed into the cell cycle with increased S phase (55.18 ± 1.38%) and G2/M phase (21.75 ± 1.38%) relative to the NC group [S-phase (13.54 ± 2.73%); G2/M phase (8.36 ± 0.28%)]. Although no significant changes were observed in the immunophenotype, the DE group showed an elevated expression of pluripotency-associated markers (OCT4, SOX2, NANOG, and REX1). These results suggest that the MFs could potentially induce proliferation of MSCs, a promising approach to promote stem cells propagation for clinical therapy and research without compromising the stemness of hUC-MSCs.

A Single-Cell Raman Spectroscopy Analysis of Bone Marrow Mesenchymal Stem/Stromal Cells to Identify Inter-Individual Diversity.

The heterogeneity of stem cells represents the main challenge in regenerative medicine development. This issue is particularly pronounced when it comes to the use of primary mesenchymal stem/stromal cells (MSCs) due to a lack of identification markers. Considering the need for additional approaches in MSCs characterization, we applied Raman spectroscopy to investigate inter-individual differences between bone marrow MSCs (BM-MSCs). Based on standard biological tests, BM-MSCs of analyzed donors fulfill all conditions for their characterization, while no donor-related specifics were observed in terms of BM-MSCs morphology, phenotype, multilineage differentiation potential, colony-forming capacity, expression of pluripotency-associated markers or proliferative capacity. However, examination of BM-MSCs at a single-cell level by Raman spectroscopy revealed that despite similar biochemical background, fine differences in the Raman spectra of BM-MSCs of each donor can be detected. After extensive principal component analysis (PCA) of Raman spectra, our study revealed the possibility of this method to diversify BM-MSCs populations, whereby the grouping of cell populations was most prominent when cell populations were analyzed in pairs. These results indicate that Raman spectroscopy, as a label-free assay, could have a huge potential in understanding stem cell heterogeneity and sorting cell populations with a similar biochemical background that can be significant for the development of personalized therapy approaches.

Generation of the human induced pluripotent stem cell line (IBKMOLi002-A) from PBMCs of a patient carrying the heterozygous L271H mutation of the voltage-gated calcium channel subunit Cav1.3-encoding CACNA1D gene

Congenital hyperinsulinemic hypoglycemia (HH) is the most frequent cause of persistent and recurrent hypoglycemia. Peripheral mononuclear blood cells (PBMCs) from a patient diagnosed with HH, alongside autism-spectrum-disorder (ASD), carrying a heterozygous c.812 T>A (L271H) mutation in the voltage-gated calcium channel subunit Cav1.3-encoding gene CACNA1D, were reprogrammed into induced pluripotent stem cells (iPSC). The CACNA1D L271H iPSC (IBKMOLi002-A) exhibit a normal karyotype, high expression of pluripotency-associated markers and the capacity to differentiate into cells of all three germ layers. We provide a novel patient-specific iPSC line, allowing to study HH, ASD, the associated neurodevelopmental disorder as well as CACNA1D-associated channelopathies in general.

Reprogramming by Cytosolic Extract of Human Embryonic Stem Cells to Improve Dopaminergic Differentiation Potential of Human Adipose Tissue-derived Stem Cells.

The extract of pluripotent stem cells induces dedifferentiation of somatic cells with restricted plasticity. In this study, we used the extract of human embryonic stem cells (hESC) to dedifferentiate adipose tissue-derived stem cells (ADSCs) and examined the impact of this reprogramming event on the dopaminergic differentiation of the cells. For this purpose, cytoplasmic extract of ESCs was prepared by repeated freezing and thawing cycles. The plasma membrane of hADSCs was reversibly permeabilized by streptolysin O (SLO), exposed to hESC extract, and resealed by a CaCl2-containing medium. As revealed by qPCR analysis, expression of OCT4, SOX2, NANOG, LIN28A, and KLF4 mRNAs were downregulated in the ADSCs one week after extract incubation, while all mRNAs except for KLF4 were upregulated at the end of the second week. For dopaminergic differentiation, control and reprogrammed ADSCs were induced by a serum-free neurobasal medium containing B27 and a cocktail of sonic hedgehog (SHH), basic fibroblast growth factor (bFGF), fibroblastic growth factor 8 (FGF8), and brain-derived neurotrophic factor (BDNF) for 12 days. After differentiation, the expression levels of some neuronal and dopaminergic-related genes, including PAX6, NESTIN, NEFL, GLI1, LMXB1, EN1, NURR1, and TH, significantly increased in the reprogrammed ADSCs compared to the control group. On the whole, two weeks after reprogramming by ESC extract, ADSCs showed an improved dopaminergic differentiation potential. These findings suggest that the cytoplasmic extract of hESCs contains some regulatory factors which induce the expression of pluripotency-associated markers in somatic cells and that the exposure to ESC extract may serve as a simple and rapid strategy to enhance the plasticity of somatic stem cells for cell replacement therapy purposes. hADSCs have emerged as a valuable candidate for transplantation therapy of neurodegenerative diseases.Several studies have documented dopaminergic dedifferentiation of hADSCs.Implementing ADSCs towards a more pluripotent state using different strategies like somatic cell nuclear transfer. The extract of pluripotent stem cells induces dedifferentiation of somatic cells with restricted plasticity. In this study, we used the extract of hESC to dedifferentiate ADSCs and examined the impact of this reprogramming event on the dopaminergic differentiation of the cells. Cytoplasmic extract of ESCs was prepared by repeated freezing and thawing cycles. These cells express several neuron-specific genes, secrete several factors associated with neuroprotection, and exhibit differentiation into neural and glial cells in vitro. In recent years, several studies have documented dopaminergic differentiation of hADSCs.

Generation and characterization of a human iPSC line HECi001-A from a healthy adult donor pancreata

Human induced pluripotent stem cells (hiPSC) are considered promising sources of pancreatic islet organoids to treat diabetes. Due to that epigenetic memory predisposes iPSCs to enhanced differentiation into the parental cell type, we sought to generate iPSC lines from islet cells isolated from healthy adult pancreata. Islet cells were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vivoteratoma formation. This cell line will be a valuable resource for drug screening, and potential source for high-efficient generation of insulin-producing islets for treating diabetes.

Generation and characterization of a human iPSC line SANi006-A from a Gray Platelet Syndrome patient

Induced pluripotent stem cells (iPSCs) were generated from erythroblasts (EBLs) obtained from a patient diagnosed with Gray Platelet Syndrome (GPS), caused by compound heterozygous NBEAL2 mutations (c.6568delT and c.7937T>C). GPS is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. EBLs were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study GPS pathophysiology and the basic functions of NBEAL2 protein in different cell types.

Generation and characterization of a human iPSC line SANi008-A from a Chédiak-Higashi Syndrome patient

Induced pluripotent stem cells (iPSCs) were generated from erythroblasts (EBLs) obtained from a patient diagnosed with Chédiak-Higashi Syndrome (CHS), caused by mutations in LYST (c.4322_4325delAGAG and c.10127A>G). EBLs were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study CHS pathophysiology and the role of LYST in different cell types.

Generation and characterization of a control and patient-derived human iPSC line containing the Hermansky Pudlak type 2 (HPS2) associated heterozygous compound mutation in AP3B1

Induced pluripotent stem cells (iPSCs) were generated from blood outgrowth endothelial cells (BOECs) obtained from a healthy donor and from a patient diagnosed with Hermansky Pudlak Syndrome type 2 (HPS2), caused by compound heterozygous AP3B1 mutations (c.177delA and c.1839-1842delTAGA). BOECs were reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study HPS2 pathophysiology and the basic functions of AP3B1 protein in different cell types.

Endothelial Cells Differentiated from Porcine Epiblast Stem Cells.

Pluripotent stem cells (PSCs) have the ability of self-renewal that can retain the characteristics of the mother cell, and of pluripotency that can differentiate into several body types. PSCs typically include embryonic stem cells (ESCs) derived from the inner cell mass of the preimplantation embryo, and epiblast stem cells (EpiSCs) derived from the epiblast of postimplantation embryo. Although PSCs are able to be used by differentiation into endothelial cells as a potential treatment for vascular diseases, human ESCs and induced PSCs (iPSCs) are followed by ethical and safety issues. Pigs are anatomically and physiologically similar to humans. Therefore, the goal of this study was to establish an efficient protocol that differentiates porcine EpiSCs (pEpiSCs) into the endothelial cells for applying the treatment of human vascular diseases. As a result, alkaline phosphatase (AP)-negative (-) pEpiSCs cultured in endothelial cell growth basal medium-2 (EBM-2) differentiation medium in association with 50 ng/mL of vascular endothelial growth factor (VEGF) for 8 days were changed morphologically like the feature of endothelial cells, and expression of pluripotency-associated markers (OCT-3/4, NANOG, SOX2, and C-MYC) in porcine differentiated cells was significantly decreased (p < 0.05). Additionally, when pEpiSCs were cultured in EBM-2 + 50 ng/mL of VEGF, porcine differentiated cells represented a common endothelial cell marker positive (CD31+) but monocytes and lymphocytes marker negative (CD45-). Therefore, these results indicated that pEpiSCs cultured in EBM-2 + 50 ng/mL of VEGF culture condition were efficiently differentiated into endothelial cells for the treatment of blood vessel diseases.

Generation of induced pluripotent stem cell (iPSC) lines carrying a heterozygous (UKWMPi002-A-1) and null mutant knockout (UKWMPi002-A-2) of Cadherin 13 associated with neurodevelopmental disorders using CRISPR/Cas9

Fibroblasts isolated from a skin biopsy of a healthy 46-year-old female were infected with Sendai virus containing the Yamanaka factors to produce transgene-free human induced pluripotent stem cells (iPSCs). CRISPR/Cas9 was used to generate isogenic cell lines with a gene dose-dependent deficiency of CDH13, a risk gene associated with neurodevelopmental and psychiatric disorders. Thereby, a heterozygous CDH13 knockout (CDH13+/-) and a CDH13 null mutant (CDH13-/-) iPSC line was obtained. All three lines showed expression of pluripotency-associated markers, the ability to differentiate into cells of the three germ layers in vitro, and a normal female karyotype.

Virus susceptibility of a new cell line derived from the fin of black carp Mylopharyngodon piceus

A continuous cell line MPF derived from the fin of black carp Mylopharyngodon piceus was established and characterised in this study. Mylopharyngodon piceus fin (MPF) cells were subcultured for more than 80 passages with high viability recovery after long-term storage. The karyotyping analysis revealed that MPF had a modal diploid chromosome number (2n = 48) and identical ribosomal RNA sequence with black carp. In addition, the expression of pluripotency-associated markers including nanog, oct4 and vasa, were detected in MPF. The transient transfection efficiency of MPF reached 23% with a fluorescent reporter by modified electroporation and stable expression of red fluorescent MPF was established by the baculovirus system, indicating that MPF is an ideal platform for studying gene functions in vitro. Lastly, cytopathic effects were also observed and RNA transcripts of a viral gene increased after infection by spring viremia of carp virus (SVCV), suggesting that MPF could be an alternative tool for investigating pathogen-host interactions in black carp. In conclusion, a fin cell line that is susceptible to SVCV was established as a potential adult stem-cell line, providing a suitable tool for future genetic analyses and pathogen-host studies in black carp.

CUL4B regulates cancer stem-like traits of prostate cancer cells by targeting BMI1 via miR200b/c.

Cancer stem-like traits contribute to prostate cancer (PCa) progression and metastasis. Cullin 4B (CUL4B) is a member of the ubiquitin E3 ligase family and overexpressed in several solid malignancies including PCa. CUL4B has been suggested to be an oncogene through epigenetic repression of tumor suppressors. However, the link between CUL4B expression and cancer stem-like phenotype remains unclear. Western blot analysis, sphere formation, and colony formation assays were used to examine the effect of CUL4B on cancer stem-like traits in PCa cells. Mechanically, bioinformatic analysis was utilized to evaluate whether BMI1 was a target of CUL4B. Moreover, real-time polymerase chain reaction, chromatin immunoprecipitation, and luciferase reporter assays were performed to identify microRNAs regulated by CUL4B. Finally, Western blot assay was used to validate the regulation of CUL4B, miR200b, and miR200c (miR200b/c) on the stem-like characteristics of PCa cells. CUL4B promotes PCa pluripotency-associated markers expression, sphere formation, and anchorage-independent growth ability in vitro. Mechanically, CUL4B upregulates BMI1 expression via epigenetically repressing miR200b/c expression. In addition, miR200b/c could partially reverse CUL4B-induced BMI1 and pluripotency-associated marker expression. Our study revealed that CUL4B regulates cancer stem-like traits of prostate cancer cells by targeting BMI1 via miR200b/c, which might give novel insight into how CUL4B promotes PCa progression through regulating cancer stem-like traits.

Fibroblast-derived integration-free iPSC line ISRM-NBS1 from an 18-year-old Nijmegen Breakage Syndrome patient carrying the homozygous NBN c.657_661del5 mutation

Human fibroblasts cells from a female diagnosed with Nijmegen Breakage Syndrome (NBS) carrying the homozygous NBN c.657_661del5 mutation were used to generate integration-free induced pluripotent stem cells (iPSCs) by over-expressing episomal-based plasmids harbouring OCT4, SOX2, NANOG, KLF4, c-MYC and LIN28. The derived iPSC line – ISRM-NBS1 was defined as pluripotent based on (i) expression of pluripotency-associated markers (ii) embryoid body-based differentiation into cell types representative of the three germ layers and (iii) the similarity between the transcriptome of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.955.

Generation of the human induced pluripotent stem cell line UKWNLi002-A from dermal fibroblasts of a woman with a heterozygous c.608 C>T (p.Thr203Met) mutation in exon 3 of the nerve growth factor gene potentially associated with hereditary sensory and autonomic neuropathy type 5

In this study, we report the human induced pluripotent stem cell line (iPSC) HSAN5-T203 M-iPSC, generated from human dermal fibroblasts (HDF) of a woman carrying a heterozygous c.608C > T mutation in the nerve growth factor gene potentially associated with hereditary sensory and autonomic neuropathy type 5 (HSAN5). HDF were reprogrammed using transient expression of key transcription factors for pluripotency and immune evasion via transfection of synthetic mRNA and miRNA. HSAN5-T203 M-iPSC retained the disease-associated genotype c.608C > T, while maintaining a normal karyotype, showed robust and abundant expression of pluripotency-associated markers and could be differentiated into cells of all three germ layers.

Previously claimed male germline stem cells from porcine testis are actually progenitor Leydig cells

BackgroundMale germline stem cells (mGSCs) offer great promise in regenerative medicine and animal breeding due to their capacity to maintain self-renewal and to transmit genetic information to the next generation following spermatogenesis. Human testis-derived embryonic stem cell-like cells have been shown to possess potential of mesenchymal progenitors, but there remains confusion about the characteristics and origin of porcine testis-derived stem cells.MethodsPorcine testis-derived stem cells were obtained from primary testicular cultures of 5-day old piglets, and selectively expanded using culture conditions for long-term culture and induction differentiation. The stem cell properties of porcine testis-derived stem cells were subsequently assessed by determining the expression of pluripotency-associated markers, alkaline phosphatase (AP) activity, and capacity for sperm and multilineage differentiation in vitro. The gene expression profile was compared via microarray analysis.ResultsWe identified two different types of testis-derived stem cells (termed as C1 and C2 here) during porcine testicular cell culture. The gene expression microarray analysis showed that the transcriptome profile of C1 and C2 differed significantly from each other. The C1 appeared to be morphologically similar to the previously described mouse mGSCs, expressed pluripotency- and germ cell-associated markers, maintained the paternal imprinted pattern of H19, displayed alkaline phosphatase activity, and could differentiate into sperm. Together, these data suggest that C1 represent the porcine mGSC population. Conversely, the C2 appeared similar to the previously described porcine mGSCs with three-dimensional morphology, abundantly expressed Leydig cell lineage and mesenchymal cell-specific markers, and could differentiate into testosterone-producing Leydig cells, suggesting that they are progenitor Leydig cells (PLCs).ConclusionCollectively, we have established the expected characteristics and markers of authentic porcine mGSCs (C1). We found for the first time that, the C2, equivalent to previously claimed porcine mGSCs, are actually progenitor Leydig cells (PLCs). These findings provide new insights into the discrepancies among previous reports and future identification and analyses of testis-derived stem cells.