Pluripotent Stem Cell Markers Research Articles

Canonical and early lineage-specific stem cell types identified in planarian SirNeoblasts

BackgroundThe pluripotent stem cells in planarians, a model for tissue and cellular regeneration, remain further identification. We recently developed a method to enrich piwi-1+ cells in Schmidtea mediterranea, by staining cells with SiR-DNA and Cell Tracker Green, named SirNeoblasts that permits their propagation and subsequent functional study in vivo. Since traditional enrichment for planarian neoblasts by Hoechst 33342 staining generates X1 cells, blocking the cell cycle and inducing cytotoxicity, this method by SiR-DNA and Cell Tracker Green represents a complementary technological advance for functional investigation of cell fate and regeneration. However, the similarities in heterogeneity of cell subtypes between SirNeoblasts and X1 remain unknown.ResultsIn this work, we performed single cell RNA sequencing of SirNeoblasts for comparison with differential expression patterns in a publicly available X1 single cell RNA sequencing data. We found first that all of the lineage-specific progenitor cells in X1 were present in comparable proportions in SirNeoblasts. In addition, SirNeoblasts contain an early muscle progenitor that is unreported in X1. Analysis of new markers for putative pluripotent stem cells identified here, with subsequent sub-clustering analysis, revealed earlier lineages of epidermal, muscular, intestinal, and pharyngeal progenitors than have been observed in X1. Using the gcm as a marker, we also identified a cell subpopulation resided in previously identified tgs-1+ neoblasts. Knockdown of gcm impaired the neoblast repopulation, suggesting a function of gcm in neoblasts.ConclusionsIn summary, the use of SirNeoblasts will enable broad experimental advances in regeneration and cell fate specification, given the possibility for propagation and transplantation of recombinant and mutagenized pluripotent stem cells that are not previously afforded to this rapid and versatile model system.

Generation of GPAM knockout human embryonic stem cell line SYSUe-008-A using CRISPR/Cas9

GPAM (glycerol-3-phosphateacyltransferase1) is a mitochondrial enzyme that catalyze an essential step in glycerolphospholipids and triacylglycerol biosynthesis process. Loss-of-function mutation of GPAM has been shown to lead to hypomyelination of corticospinal tract in cerebral palsy patient. To model this rare disease with human brain organoid, we generated a GPAM knockout human embryonic stem cell line SYSUe-008-A by CRISPR/cas9. The GPAM knockout cell line maintains a normal karyotype and shows comparable level of pluripotent stem cell marker expression and differentiation potential as wild-type human embryonic stem cells.

Expression of SSEA-4 and Oct-4 from somatic cells in primary mouse gastric cell culture induced by brief strong acid.

Environmental changes can stress and alter biology at the molecular and cellular level. For example, metal-protein interaction is a classic physic and biological property of nature, which is fundamentally influenced by acidity. Here, we report a unique cellular reprogramming phenomenon in that a brief strong acid treatment induced the expression of pluripotent stem cell (PSC) markers. We used strong acid to briefly challenge mix-cultured gastric cells, and then subcultured survived cells in anormal cell culture medium. We found that survival acid-treated cells expressed PSC markers detected by commonly used pluripotent antibodies such as SSEA-4 and Oct4. In addition, we observed that the survived cells from theacid challenge grew faster during the second and third weeks of subculture and hada relative short doubling time (DT) than the controls. PSC marker-labeled 'older' cells also presented immature cell-like morphology with some having marker Oct4 in the nucleus. Finally, the expression of the markers appeared to be sensitive to metal ion chelation. Removal of the metals during a brief acid treatment reduced pluripotent marker-positive cells, suggesting the dissociation of metals from metal-binding proteins may be a factor involved in the induction of stem cell markers. Our findings reveal that somatic cells appear to possess a plasticity feature to express pluripotent marker proteins or to select cell subpopulations that express pluripotent marker proteins when cells are transiently exposed to strong acid. It opens new directions for understanding conserved regulatory mechanisms involved in cellular survival under stressful stimulation.

Generation of PARP1 gene knockout human embryonic stem cell line using CRISPR/Cas9

PARP1 encodes a chromatin-associated enzyme which responsible for post-translational poly(ADP-ribosyl)ation modification (Hsieh et al., 2017). It plays an important role in nucleotide excision repair, non-homologous end joining, DNA mismatch repair and many other DNA repair process. Also, PARP1 participates in inflammation and aging. However, its role in human embryonic stem cell biology has not been fully resolved. To clarify the function of PARP1 in human embryonic stem cells, we reported a PARP1 knockout human embryonic stem cell line, generated by CRISPR/Cas9 mediated gene targeting. This cell line shows normal karyotype, pluripotent stem cell marker expression and differentiation potential in vitro.

Embryonic Stem Cell-like Population in Hypertrophic Port-wine Stain

Objective: To identify and characterize an embryonic stem cell (ESC)-like population within hypertrophic port-wine stain (HPWS). Methods: HPWS tissue samples from 15 patients underwent immunohistochemical staining for induced pluripotent stem cell (iPSC) markers OCT4, SOX2, NANOG, KLF4, and c-MYC. Immunofluorescence staining was performed on 2 of these tissue samples to investigate colocalization of these markers. In situ hybridization and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) were performed on 6 of the HPWS samples to investigate transcript expression of these iPSC markers. Western blotting and RT-qPCR were performed on 3 HPWS-derived primary cell lines, to determine protein and transcript expression of these markers, respectively. Results: Immunohistochemical staining demonstrated expression of OCT4, SOX2, KLF4, and c-MYC but not NANOG on the endothelium and media of lesional vessels and on cells within the stroma in all 15 HPWS tissue samples. Immunofluorescence staining showed the presence of an OCT4+/SOX2+/NANOG-/KLF4+/c-MYC+ ESC-like subpopulation within the endothelium and media of the lesional vessels, and cells within the stroma of HPWS. In situ hybridization detected OCT4, SOX2, KLF4, and c-MYC transcripts in all 6 HPWS tissue samples. RT-qPCR demonstrated transcripts of all 5 iPSC markers in the HPWS tissue samples and in the HPWS-derived primary cell lines, which expressed OCT4, SOX2, KLF4, and c-MYC but not NANOG proteins by Western blotting. Conclusion: This study demonstrated an OCT4+/SOX2+/NANOG-/KLF4+/c-MYC+ ESC-like population within the endothelium and media of the lesional vessels and the cells within the stroma of HPWS.

Gel-Free 3D Tumoroids with Stem Cell Properties Modeling Drug Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer

Researchers have developed several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids with increased properties of cancer stem cells (CSCs), also called cancer-initiating cells (CICs). Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anti-cancer drugs have been reported using 2D culture systems, whereas 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aimed to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyrosine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter the tumoroid growth of metastatic colorectal cancer (mCRC). Gene expression signatures of highly metastatic aggregative CRC (LuM1 cells) vs. low-metastatic, non-aggregative CRC (Colon26 and NM11 cells) were analyzed using microarray. To establish a 3D culture-based multiplexing reporter assay system, LuM1 was stably transfected with the Mmp9 promoter-driven ZsGreen fluorescence reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate®, a gel-free 3D culture device. LuM1 cells highly expressed mRNA encoding ABCG2 (a drug resistance pump, i.e., CSC/CIC marker), other CSC/CIC markers (DLL1, EpCAM, podoplanin, STAT3/5), pluripotent stem cell markers (Sox4/7, N-myc, GATA3, Nanog), and metastatic markers (MMPs, Integrins, EGFR), compared to the other two cell types. Hoechst efflux stem cell-like side population was increased in LuM1 (7.8%) compared with Colon26 (2.9%), both of which were markedly reduced by verapamil treatment, an ABCG2 inhibitor. Smaller cell aggregates of LuM1 were more sensitive to Cisplatin (at 10 μM), whereas larger tumoroids with increased ABCG2 expression were insensitive. Notably, Cisplatin (2 μM) and Imatinib (10 μM) at low concentrations significantly promoted tumoroid formation (cell aggregation) and increased Mmp9 promoter activity in mCRC LuM1/m9, while not cytotoxic to them. On the other hand, 5-FU significantly inhibited tumoroid growth, although not completely. Thus, drug resistance in cancer with increased stem cell properties was modeled using the gel-free 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance.

Characterization of stem cells from human ovarian follicular fluid; a potential source of autologous stem cell for cell-based therapy.

Human ovarian follicular fluid (HOFF) contains proteins, extracellular matrixes necessary for growth and maturation of oocytes as well as granulosa cells. Epithelial cells and stem cells can be isolated from HOFF. However, information regarding stem cells derived from HOFF is still lacking. The objectives of the present study were to isolate, characterize, and differentiate cells derived from HOFF. HOFF was collected during the routine aspiration of oocytes in an assisted fertilization program and subjected to cell isolation, characterization, and in vitro culture. After 24h of culture, different cell morphologies including epithelial-like-, neural-like- and fibroblast-like cells were observed. Immunocytochemistry reveals the expression of pluripotent stem cell markers (OCT4, NANOG, SSEA4), epithelial marker (CK18), FSH- and LH-receptor. For in vitro culture, the isolated cells were continuously cultured in a growth medium; alpha MEM containing 10% FBS and epidermal growth factor (EGF). After 2weeks of in vitro culture, cells with fibroblast-like morphology dominantly grow in the culture vessels and resemble mesenchymal stem cells (MSCs). HOFF-derived cells exhibited MSC expression of CD44, CD73, CD90, CD105, CD146, and STRO-1, and were capable of differentiation into osteoblasts, chondrocytes, and adipocytes. After induction of neural differentiation, HOFF-derived cells formed spheroidal structures and expressed neural stem cell markers including Nestin, β-tubulin III, and O4. Besides, the oocyte-like structure was observed after prolonged culture of HOFF. In conclusion, cells derived from follicular fluidexhibited stem cell characteristics, which could be useful for regenerative medicine applications and cell-based therapies.

Establishment and characterization of induced pluripotent stem cell line (IGIBi002-A) from a β-thalassemia patient with IVS1-5 mutation by non-integrating reprogramming approach.

β-thalassemia (BT) is a hereditary blood disorder caused by mutations in the β-globin (HBB) gene leading to severely reduced or no synthesis of the β-chain of adult hemoglobin. IVS1-5 (G>C) is the most common BT mutation in Indian population and yet no patient-specific cellular models have been generated. Here, we have established an induced pluripotent stem cell (iPSC) line, IGIBi002-A from a thalassemia patient with a homozygous IVS1-5(G>C) mutation. Characterization of IGIBi002-A demonstrated that these iPSCs are free of exogenous reprogramming genes and expressed pluripotent stem cell markers, exhibited a normal karyotype and were potential of three germ layer differentiation.

Generation of GADD45A gene knockout human embryonic stem cell line using CRISPR/Cas9.

GADD45A is a DNA damage and stressful growth arrest inducible protein, also it is shown to a be tumor suppressor gene and a chromatin relaxer associated with opening chromatin during the somatic reprogramming. However, its role in human embryonic stem cells and human embryonic stem cell modeled development has been merely documented. To illustrate the function of GADD45A in the human embryonic stem cell biology, we reported a GADD45A knockout human embryonic stem cell line by CRISPR/Cas9 mediated gene targeting. This cell line displayed normal karyotype, pluripotent stem cell marker expression and differentiation potential both in vivo and vitro.

Generation of a homozygous CRISPR/Cas9-mediated knockout H9 hESC subline for the CRB1 locus.

Mutations in the CRB1 gene reportedly cause early-onset autosomal recessive retinitis pigmentosa (RP), which can result in severe loss of vision at an early age. To investigate the mechanism of CRB1-knockout (CRB1-/-) induced RP, we generated a subline of H9 human embryonic stem cells harboring frame shift mutations in a homozygous state in exon 2 of the CRB1 gene. This subline expressed pluripotent stem cell markers, presented a normal karyotype, and preserved the ability to differentiate into endodermal, mesodermal, and ectodermal lineages.

The Clinical and Prognostic Implications of Pluripotent Stem Cell Markers Expression and Their Correlation with the WNT signal pathway in Hepatocellular Carcinoma.

Objectives:This study aimed to investigate the expression of SOX2, SOX9, p53, and β-catenin in hepatocellular carcinoma (HCC) and their correlation with clinicopathological parameters of prognostic importance. Materials and Methods:Seventy-five patients were enrolled in this study. All patients had full clinical and follow-up data and available paraffin blocks. Immunohistochemical analysis was performed and correlated with clinicopathological factors and patient survival. Results:We detected the positive expression of SOX2, SOX9, p53, and β-catenin in 76%, 50.7%, 50.7%, and 77.9% of HCC specimens respectively. All studied markers showed a significant increase in the expression in tumor tissue specimens compared to non-tumor tissue. Both SOX2 and SOX9 expressions were significantly associated with adverse prognostic factors in HCC. Significant positive correlations were found between SOX2 and SOX9 and both p53 and β-catenin expression (r= 0.528, 0.485 and; r = 0.253, 0.327, respectively; p< 0.0001 for both of them). Regarding survival, we found that HCC patients with positive SOX2 and SOX9 expressions had significantly shorter overall survival (p=0.0001, each). Additionally, larger tumor size, tumor grade, high stage, tumor multiplicity, presence of cirrhosis, tumor necrosis, high p53 expression, and positive β-catenin expression were independent predictors of worse survival. A multivariate Cox analysis revealed that tumor grade, stage, p53, and SOX2 expression were independent predictors of unfavorable prognosis in overall survival (p=0.0001, p=0.0001,p=0.033; and p=0.003, respectively). Conclusions:Our findings might provide an insight into SOX2 and SOX9’s role in HCC and suggest that SOX2 might be targeted for HCC therapy.

Heterogeneity and hierarchy of the tissue stem cells in the human newborn vocal fold mucosa.

ObjectivesThere is growing evidence that the cells in the maculae flavae (MFe) are tissue stem cells and the MFe are a stem cell niche of the human vocal fold mucosa. Heterogeneity and hierarchy of tissue stem cells in the MFe of newborn vocal fold were investigated in vivo.Study designHistologic analysis of the human vocal folds.MethodsFive normal human newborn vocal folds were investigated under transmission electron microscopy and light microscopy.ResultsCobblestone‐like polygonal cells, vocal fold stellate cell‐like cells, and fibroblast‐like spindle cells were intermingled in the newborn MFe in vivo, indicating that the cells in the MFe had heterogeneity. However, cobblestone‐like polygonal cells were predominant. Free ribosomes were well developed in the cytoplasm. The cells in some cases formed gap junctions with each other. The cells in some cases were attached to other cells and formed cell junctions with each other. These findings indicated cells in the newborn maculae flavae possessed features of mesenchymal cells (cells in mesenchyme). Colony‐forming‐unit‐like cell aggregate was observed, indicating the cells in the newborn MFe had stemness. The cobblestone‐like polygonal cells expressed SSEA‐3 (a human pluripotent stem cell marker), indicating they were at the top of a cellular hierarchy in the stem cell system.ConclusionsThe cells in the MFe of the human newborn vocal fold mucosa had heterogeneity and hierarchy in the stem cell system in vivo. At birth, newborn maculae flavae are ready to start the growth of the vocal fold mucosa as a vibrating tissue.

Generation of 10 patient-specific induced pluripotent stem cells (iPSCs) to model Pitt-Hopkins Syndrome.

Autosomal dominant mutations in transcription factor 4 (TCF4) are associated with a rare syndromic form of Autism Spectrum Disorder (ASD) called Pitt-Hopkins Syndrome (PTHS). Here, we report the generation of a collection of induced pluripotent stem cells (iPSCs) from 5 patients diagnosed with PTHS and 5 familial controls. These patient-derived iPSCs contain a variety of mutations within the TCF4 gene, possess a normal karyotype and express all the appropriate pluripotent stem cell markers. These novel patient lines will be a useful resource for the research community to study PTHS and the function of TCF4.

Establishment of a human induced pluripotent stem cell line, JUCTCi012-A, from multiple symmetric lipomatosis (MSL) patient carrying a homozygous Arg707Trp (c.2119C>T) mutation in the MFN2 gene.

Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts collected from a 39-year-old multiple symmetric lipomatosis (MLS) female patient carrying a point mutation in MFN2 gene (c.2119C>T). The resulting iPSCs showed typical embryonic-like morphology, expressed pluripotency stem cell markers, retained the normal karyotype after reprogramming and showed the potential to differentiate into three germ layers. This iPSC line can be used for studying MSL disease mechanisms.

Dermatan sulphate promotes neuronal differentiation in mouse and human stem cells.

Dermatan sulphate (DS), a glycosaminoglycan, is present in the extracellular matrix and on the cell surface. Previously, we showed that heparan sulphate plays a key role in the maintenance of the undifferentiated state in mouse embryonic stem cells (mESCs) and in the regulation of their differentiation. Chondroitin sulphate has also been to be important for pluripotency and differentiation of mESCs. Keratan sulphate is a marker of human pluripotent stem cells. To date, however, the function of DS in mESCs has not been clarified. Dermatan 4 sulfotransferase 1, which transfers sulphate to the C-4 hydroxyl group of N-acetylgalactosamine of DS, contributes to neuronal differentiation of mouse neural progenitor cells. Therefore, we anticipated that neuronal differentiation would be induced in mESCs in culture by the addition of DS. To test this expectation, we investigated neuronal differentiation in mESCs and human neural stem cells (hNSCs) cultures containing DS. In mESCs, DS promoted neuronal differentiation by activation of extracellular signal-regulated kinase 1/2 and also accelerated neurite outgrowth. In hNSCs, DS promoted neuronal differentiation and neuronal migration, but not neurite outgrowth. Thus, DS promotes neuronal differentiation in both mouse and human stem cells, suggesting that it offers a novel method for efficiently inducing neuronal differentiation.

Cancer Stem Cell Subpopulations Are Present Within Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma.

Cancer stem cells (CSCs) have been identified in many cancer types including primary head and neck cutaneous squamous cell carcinoma (HNcSCC). This study aimed to identify and characterize CSCs in metastatic HNcSCC (mHNcSCC). Immunohistochemical staining performed on mHNcSCC samples from 15 patients demonstrated expression of the induced pluripotent stem cell (iPSC) markers OCT4, SOX2, NANOG, KLF4, and c-MYC in all 15 samples. In situ hybridization and RT-qPCR performed on four of these mHNcSCC tissue samples confirmed transcript expression of all five iPSC markers. Immunofluorescence staining performed on three of these mHNcSCC samples demonstrated expression of c-MYC on cells within the tumor nests (TNs) and the peri-tumoral stroma (PTS) that also expressed KLF4. OCT4 was expressed on the SOX2+/NANOG+/KLF4+ cells within the TNs, and the SOX2+/NANOG+/KLF4+ cells within the PTS. RT-qPCR demonstrated transcript expression of all five iPSC markers in all three mHNcSCC-derived primary cell lines, except for SOX2 in one cell line. Western blotting showed the presence of SOX2, KLF4, and c-MYC but not OCT4 and NANOG in the three mHNcSCC-derived primary cell lines. All three cell lines formed tumorspheres, at the first passage. We demonstrated an OCT4+/NANOG+/SOX2+/KLF4+/c-MYC+ CSC subpopulation and an OCT4+/NANOG-/SOX2+/KLF4+/c-MYC+ subpopulation within the TNs, and an OCT4+/NANOG+/SOX2+/KLF4+/c-MYC+ subpopulation within the PTS of mHNcSCC.

Derivation and 97% Purification of Human Thyroid Cells From Dermal Fibroblasts.

Background: The success in rescuing thyroid deficiency in mice using thyroid cells derived from embryonic stem (ES) cells, together with the discovery of human induced pluripotent stem cells (iPSCs) from somatic cells, has raised the possibility of patient-specific thyroid cell replacement. In this study we demonstrate that human thyroid follicular cells can be derived from human iPSCs and show the ability of highly purified and differentiated cells to secrete thyroid hormone.Research Design and Methods: Human iPSCs were derived from adult skin fibroblasts using RNA reprogramming and differentiated in vitro into thyroid follicular cells by exposure to activin A, ethacridine and TSH as we have previously described for human ES cells. The resulting thyroid cells were then highly purified using double antibody cell sorting.Results: The iPSCs derived from human dermal fibroblasts showed stem cell-like morphologic changes and expressed pluripotent stem cell markers as assessed using qPCR, immunofluorescence staining, and FACS analysis. These cells retained their pluripotential characteristics as shown by teratoma formation after murine transplantation. Definitive endoderm cells were induced with activin A and the transcription factor TAZ was significantly induced on ethacridine treatment and translocated to the nucleus. Thyroid transcription factors NKX2-1 and PAX8 were also highly expressed in activin A derived endoderm cells and further induced by ethacridine. Following terminal differentiation with TSH, there was enhanced thyroid follicle formation, high expression of the thyroid specific genes—TG, TPO, TSHR and NIS, and secretion of thyroid hormone (T4) in vitro. Furthermore, we were able to achieve a 97% purification of TSHR+/NIS+ expressing cells after differentiation using a single purification procedure.Conclusions: These findings demonstrate that mature adult dermal fibroblasts can be matured into human iPSCs which have the potential to form functional thyroid follicular cells. This lays the groundwork for future person–specific thyroid regenerative therapy.

Establishment of (JUCTCi007-A) iPSC line from a patient with congenital myasthenic syndrome (CMS) carrying a homozygous mutation p.Arg331Trp (c.991C>T) in the CHRNE gene.

Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts obtained from a 24-year-old female diagnosed with hereditary congenital myasthenic syndrome (CMS), caused by p.Arg331Trp (c.991C>T) homozygous mutation in the gene coding for the epsilon subunit of the acetylcholine receptor (CHRNE). The generated iPSCs shared similar karyotype with the parental dermal fibroblast cells, expressed pluripotency stem cell markers, and demonstrated differentiation potential into the three germ layers. This cell line can be used as an ideal model to facilitate the understanding of the pathogenic disease mechanisms underlying the congenital myasthenic syndrome and for developing novel therapeutic strategies.

Generation of an ALS human iPSC line KEIOi001-A from peripheral blood of a Charcot disease-affected patient carrying TARDBP p.N345K heterozygous SNP mutation.

Amyotrophic Lateral Sclerosis is the most common motor neuron degenerative disease in adults, and TARDBP gene mutations have been reported to be involved in the pathogenesis. We present here how we generated the human induced pluripotent stem cell (hiPSC) line KEIOi001-A/SM4-4-5 from the peripheral blood of a 63-year-old male patient presenting the c.1035C>G heterozygous SNP mutation in the TARDBP gene locus. The established hiPSC line does not express the exogenous reprogramming factors oriP nor EBNA1 and shows no karyotypic abnormalities, while it expresses pluripotent stem cell markers, presents the SNP mutation and is capable of three-germ layers differentiation in vitro.

Establishment of a human induced pluripotent stem cell (iPSC) line (JUCTCi010-A) from a healthy Jordanian female skin dermal fibroblasts.

Human integration-free induced pluripotent pluripotent stem cells (hiPSCs) were generated from skin fibroblasts obtained from a 27-year-old healthy Jordanian female. The resulting iPSCs expressed the most common pluripotency stem cell markers, they retained the normal karyotype similar to the original fibroblasts and showed the potential to differentiate into three germ layers in vitro. This iPSC line could serve as a wild-type control that can be used in hereditary disease modeling studies and in optimization of different differentiation protocols.