Embryonic Stem Cell Markers Research Articles

Human Second Trimester Amniotic Fluid Cells are Able to Create Embryoid Body-Like Structures in Vitro and to Show Typical Expression Profiles of Embryonic and Primordial Germ Cells

Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

BMP-2 inhibits tumor-initiating ability in human renal cancer stem cells and induces bone formation.

We have previously shown that BMP-2 induces bone formation and inhibits tumorigenicity of cancer stem cells (CSCs) in a human osteosarcoma OS99-1 cell line. In this study, we sought to determine whether BMP-2 can similarly induce bone formation and inhibit the tumorigenicity of renal CSCs identified based on aldehyde dehydrogenase (ALDH) activity in renal cell carcinoma (RCC) cell lines and primary tumors. Using a xenograft model in which cells from human RCC cell lines ACHN, Caki-2, and primary tumors were grown in NOD/SCID mice, renal CSCs were identified as a subset of ALDH(br) cells. The ALDH(br) cells possessed a greater colony-forming efficiency, higher proliferative output, increased expression of stem cell marker genes Oct3/4A, Nanog, renal embryonic marker Pax-2, and greater tumorigenicity compared to cells with low ALDH activity (ALDH(lo) cells), generating new tumors with as few as 25 cells in mice. In vitro, BMP-2 was found to inhibit the ALDH(br) cell growth, down-regulate the expression of embryonic stem cell markers, and up-regulate the transcription of osteogenic markers. In vivo, all animals receiving a low number of ALDH(br) cells (5 × 10(3)) from ACHN, Caki-2, and primary tumor xenografts treated with 30 µg BMP-2 per animal showed limited tumor growth with significant bone formation, while untreated cells developed large tumor masses without bone formation. These results suggest that BMP-2 inhibits the tumor-initiating ability of renal CSCs and induces osseous bone formation. BMP-2 may therefore provide a beneficial strategy for human RCC treatment by targeting the CSC-enriched population.

Characteristics of stem cells derived from rat fascia: in vitro proliferative and multilineage potential assessment.

Fascia‑derived stem cells (FDSCs) were previously isolated from the fascia of the gluteus maximus of the rat. However, the use of FDSCs as a cell source for musculoskeletal tissue engineering has not been compared with that of adipose‑derived stem cells (ADSCs) and bone marrow‑derived mesenchymal stem cells (BMSCs). Therefore, the present study aimed to compare the mesenchymal stem cell (MSC) and self‑renewal stem cell markers, proliferative capacity and multilineage differentiation potential of these stem cells in vitro. The MSC and embryonic stem cell (ESC) marker profiles were compared using flow cytometry and quantitative polymerase chain reaction (qPCR). Their proliferative capacities were compared using 5‑bromo‑2'‑deoxyuridine and MTT assays. Their osteogenic, adipogenic and chondrogenic differentiation potentials were compared using standard staining assays and qPCR. The FDSCs possessed similar cell morphology and immunophenotypic profiles with BMSCs and ADSCs. FDSCs demonstrated a similar expression pattern of ESC markers with ADSCs, which has higher expression of sex determining region Y‑box (Sox)2 and octamer‑binding transcription factor 4, and lower expression of Krüppel‑like factor 4, when compared with BMSCs. FDSCs exhibited higher proliferation under serum‑deprived conditions (0.5% FBS growth medium), and attained higher expression levels of collagen type I, α 2 and type II, α 1 as well as Sox9 mRNA than ADSCs and BMSCs upon chondrogenic induction. An increased amount of proteoglycan deposition was also observed in the FDSC group. However, lower levels of adipogenic and osteogenic marker expression in FDSCs were detected compared with ADSCs and BMSCs upon adipogenic and osteogenic induction, respectively. FDSCs possessed high chondrogenic potential, low osteogenic and adipogenic differentiation potential and were responsive to the induction signals for collagen‑rich fascial structure regeneration. Therefore, FDSCs may represent an improved alternative cell source to conventional ADSCs and BMSCs for musculoskeletal tissue repair and tissue engineering, particularly for collagen‑rich structures with poor vasculature.

Profiling gene promoter occupancy of Sox2 in two phenotypically distinct breast cancer cell subsets using chromatin immunoprecipitation and genome-wide promoter microarrays.

IntroductionAberrant expression of the embryonic stem cell marker Sox2 has been reported in breast cancer (BC). We previously identified two phenotypically distinct BC cell subsets separated based on their differential response to a Sox2 transcription activity reporter, namely the reporter-unresponsive (RU) and the more tumorigenic reporter-responsive (RR) cells. We hypothesized that Sox2, as a transcription factor, contributes to their phenotypic differences by mediating differential gene expression in these two cell subsets.MethodsWe used chromatin immunoprecipitation and a human genome-wide promoter microarray (ChIP-chip) to determine the promoter occupancies of Sox2 in the MCF7 RU and RR breast cancer cell populations. We validated our findings with conventional chromatin immunoprecipitation, quantitative reverse transcription polymerase chain reaction (qPCR), and western blotting using cell lines, and also performed qPCR using patient RU and RR samples.ResultsWe found a largely mutually exclusive profile of gene promoters bound by Sox2 between RU and RR cells derived from MCF7 (1830 and 456 genes, respectively, with only 62 overlapping genes). Sox2 was bound to stem cell- and cancer-associated genes in RR cells. Using quantitative RT-PCR, we confirmed that 15 such genes, including PROM1 (CD133), BMI1, GPR49 (LGR5), and MUC15, were expressed significantly higher in RR cells. Using siRNA knockdown or enforced expression of Sox2, we found that Sox2 directly contributes to the higher expression of these genes in RR cells. Mucin-15, a novel Sox2 downstream target in BC, contributes to the mammosphere formation of BC cells. Parallel findings were observed in the RU and RR cells derived from patient samples.ConclusionsIn conclusion, our data supports the model that the Sox2 induces differential gene expression in the two distinct cell subsets in BC, and contributes to their phenotypic differences.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0470-2) contains supplementary material, which is available to authorized users.

Immunoexpressions of embryonic and nonembryonic stem cell markers (Nanog, Thy-1, c-kit) and cellular connections (connexin 43 and occludin) on testicular tissue in thyrotoxicosis rat model

In this study, possible thyrotoxicosis-related histological changes in testicular tissues of rats with experimentally induced thyrotoxicosis model were evaluated on cellular connections and stem cell markers. Two experimental groups, thyrotoxicosis and control, each consisting of eight animals were used. Rats in the thyrotoxicosis group were injected intraperitoneally with 3,3',5-triiodo-l-thyronine (50 µg/100 g body weight/day) for 10 days. At the end of the study, animals in both groups were anesthetized, and blood samples were collected for biochemical analyses. Their testes were dissected out and histological procedure was conducted to perform further histochemical, immunohistochemical analyses and tissue expression analysis by real-time polymerase chain reaction. Expression of the stem cell markers such as c-kit and Thy-1 significantly decreased in the testes of the thyrotoxicosis group compared with the control group; however, Nanog expression was not detected in any of the groups. Similarly, connexin 43 and occludin expressions were also found to be significantly lower in the thyrotoxicosis group. These results on cellular connections are supported with the tissue expression analysis. Our findings are indicative of supporting microenvironmental tissue decay rather than parenchyma damage, which has been actually ignored in the literature. In conclusion, experimental thyrotoxicosis model may have adverse effects on the cell junctional complexes, cell-cell interactions, and pluripotency capacity.

Cancer stem cells are enriched in Fanconi anemia head and neck squamous cell carcinomas.

Fanconi anemia (FA) patients have an increased risk of head and neck squamous cell carcinoma (HNSCC) at a higher rate with no apparent risk factors. HNSCC of FA patients is an aggressive tumor characterized by multifocal origin, early metastases and frequent recurrences. Given that cancer stem cells (CSC) drive tumorigenesis, tumor recurrence and metastasis, in this study, we characterized the CSC population in FA and sporadic HNSCC. The Aldefluor assay was used to characterize and isolate CSC with high aldehyde dehydrogenase (ALDH) activity (ALDHpos) in cell lines derived from FA and sporadic HNSCC. Isolated ALDHpos and ALDHneg cells were examined for the expression of stemness genes using reverse transcription-polymerase chain reaction (RT-PCR) array. Tumor cell-derived FA and sporadic HNSCC were examined for their ability to form tumorspheres in vitro. Stem-like cell population in FA and sporadic HNSCC in human and mouse xenograft tumors were evaluated using ALDH isoform 1 (ALDH1) immunohistochemistry. FA-HNSCC cell lines harbor a greater proportion of ALDHpos cells (15–31%) compared to sporadic HNSCC (10%). Expression of Nanog, Oct-3/4 and Stella, molecular markers of undifferentiated embryonic stem (ES) cells were detected in the ALDHpos FA-HNSCC cells and not in the ALDHneg cells. FA-HNSCC cell lines revealed enhanced in vitro tumorsphere formation compared to sporadic HNSCC cells. A higher percentage of ALDH1pos tumor cells are noted in the human and mouse xenograft tumors of FA-HNSCC compared to sporadic HNSCC tumors. FA-HNSCC are highly enriched for CSC and may serve as a model to develop CSC-targeted therapies for HNSCC.

Modified protocol for improvement of differentiation potential of menstrual blood-derived stem cells into adipogenic lineage.

To characterize potency of menstrual blood-derived stem cells (MenSCs) for future cell therapies, we examined differentiation potential of MenSCs into adipocytes. Differentiation potential of MenSCs in comparison to bone marrow stem cells (BMSCs) was assessed in conventional culture medium. Differentiation potential of MenSCs into adipocytes was improved using different combinations of growth factors and hormones. First, we demonstrated that MenSCs preserve their appearance and karyotypic stability during passages. Although these cells express mesenchymal stem cells markers, they cannot simply be classified as mesenchymal stem cells due to expression of embryonic stem cells marker, OCT-4. Oil red O staining showed that differentiated MenSCs in conventional medium with/without retinoic acid (protocols 1 and 2) did not attain adipocyte characteristics, whereas differentiated BMSCs in conventional medium accumulated oil vacuoles typically. Nevertheless, real-time RT-PCR results showed that LPL gene expression was up-regulated in both protocols 1 and 2, whereas LEPR was up-regulated only in protocol 2 (fortified with retinoic acid). Surprisingly, protocol 3 (including rosiglitazone) had odd influence on mRNA expression of all genes (LEPR, LPL and PPAR-γ). Oil red O staining confirmed fat-producing ability of MenSCs under protocol 3. Presented data suggest an efficient differentiation protocol for in vitro production of MenSC-derived adipocytes. These cells are suggested to be an apt alternative to BMSCs for future stem cell therapy of soft tissue injuries.

Semaphorin 3A Induces Mesenchymal-Stem-Like Properties in Human Periodontal Ligament Cells

Periodontal ligament stem cells (PDLSCs) have recently been proposed as a novel option in periodontal regenerative therapy. However, one of the issues is the difficulty of stably generating PDLSCs because of the variation of stem cell potential between donors. Here, we show that Semaphorin 3A (Sema3A) can induce mesenchymal-stem-like properties in human periodontal ligament (PDL) cells. Sema3A expression was specifically observed in the dental follicle during tooth development and in parts of mature PDL tissue in rodent tooth and periodontal tissue. Sema3A expression levels were found to be higher in multipotential human PDL cell clones compared with low-differentiation potential clones. Sema3A-overexpressing PDL cells exhibited an enhanced capacity to differentiate into both functional osteoblasts and adipocytes. Moreover, PDL cells treated with Sema3A only at the initiation of culture stimulated osteogenesis, while Sema3A treatment throughout the culture had no effect on osteogenic differentiation. Finally, Sema3A-overexpressing PDL cells upregulated the expression of embryonic stem cell markers (NANOG, OCT4, and E-cadherin) and mesenchymal stem cell markers (CD73, CD90, CD105, CD146, and CD166), and Sema3A promoted cell division activity of PDL cells. These results suggest that Sema3A may possess the function to convert PDL cells into mesenchymal-stem-like cells.

Elevated O-GlcNAc levels activate epigenetically repressed genes and delay mouse ESC differentiation without affecting naïve to primed cell transition.

The differentiation of mouse embryonic stem cells (ESCs) is controlled by the interaction of multiple signaling pathways, typically mediated by post‐translational protein modifications. The addition of O‐linked N‐acetylglucosamine (O‐GlcNAc) to serine and threonine residues of nuclear and cytoplasmic proteins is one such modification (O‐GlcNAcylation), whose function in ESCs is only now beginning to be elucidated. Here, we demonstrate that the specific inhibition of O‐GlcNAc hydrolase (Oga) causes increased levels of protein O‐GlcNAcylation and impairs differentiation of mouse ESCs both in serum‐free monolayer and in embryoid bodies (EBs). Use of reporter cell lines demonstrates that Oga inhibition leads to a reduction in the number of Sox1‐expressing neural progenitors generated following induction of neural differentiation as well as maintained expression of the ESC marker Oct4 (Pou5f1). In EBs, expression of mesodermal and endodermal markers is also delayed. However, the transition of naïve cells to primed pluripotency indicated by Rex1 (Zfp42), Nanog, Esrrb, and Dppa3 downregulation and Fgf5 upregulation remains unchanged. Finally, we demonstrate that increased O‐GlcNAcylation results in upregulation of genes normally epigenetically silenced in ESCs, supporting the emerging role for this protein modification in the regulation of histone modifications and DNA methylation. Stem Cells 2014;32:2605–2615

P17.07 * FENOFIBRATE AS A NOVEL THERAPEUTIC ADJUVANT FOR TARGETING GLIOMA STEM CELLS

Glioma stem cells (GSC) are being increasingly recognized as the culprits behind the resistance of glioblastoma to standard therapy, including surgery, temozolomide and radiation therapy. Intensive research is currently underway to uncover novel and effective GSC targeting strategies Fenofibrate is an FDA-approved hypolipidemic agent in widespread clinical use. Recent evidence has supported a possible role for fenofibrate as an nti-cancer agent in various cancer pathologies, including glioblastoma. Our own previous work has demonstrated that fenofibrate reduces proliferation and induces apoptosis in human high-grade glioma cells. Here, we present our results on the effects of fenofibrate on GSC in the in vitro setting. We chose to examine invasion because this property is known as a hallmark obstacle in the successful treatment of glioblastoma and recent data has implicated GSC. We found that fenofibrate treatment at a dose of 25 microM significantly decreased GSC invasion in a standard Matrigel invasion assay. We then examined the effect of fenofibrate on stem-cell marker expression and chose CD133 and Oct4. CD133 remains one of the most commonly used GSC markers to date, while Oct4 is an embryonic stem cell marker linked to glioma grade. Results demonstrated that fenofibrate treatment at 25 microM significantly diminished expression of both CD133 and Oct4 as measured by flow cytometry and/or immunofluorescence staining. Finally, we investigated possible mechanisms and identified NF-kappaB and CyclinD1 as candidates. Inhibition of NF-kappaB reportedly decreases invasion in GSC and is a well-established property of fenofibrate in the inflammatory setting. Downregulation of Cyclin D1 has been found to correlate with attenuated invasion in human glioblastoma cells and may be observed with fenofibrate treatment of lymphoma cells. Using a panel of established human glioma cell lines, we found that fenofibrate treatment robustly diminished the expression of both NF-kappaB and Cyclin D1 in a dose-dependent and p53-independent manner. In conclusion, our data show that fenofibrate significantly attenuates both GSC invasion and expression of the CD133 and Oct4 stem-cell markers, in association with diminished NF-kappaB and Cyclin D1 signaling in glioma cells. Based on these results, we propose that fenofibrate should be considered a novel and direct GSC targeting agent. Its relatively low cost, minimal toxicity and availability make it an attractive agent for rapid translation to the clinical setting as an adjuvant treatment for glioblastoma.

C-KIT-positive undifferentiated tumor of the liver: A case report.

With recent advances in cancer stem cell analysis, it has been postulated that the transformation of hepatic stem and progenitor cells underlies the development of certain liver cancers. Human C-KIT is a transmembrane type III receptor protein with intrinsic tyrosine kinase activity that has been proposed as a marker for human embryonic stem cells. In addition, human C-KIT functions in maintaining the undifferentiated state of stem cells, and has been identified as a marker for human hematopoietic and hepatic stem/progenitor cells. The present study identified an unusual case of a C-KIT-positive hepatic tumor with an undifferentiated stem cell phenotype distinct from existing descriptions of liver tumors. A 69-year-old male with Ampulla of Vater (AoV) cancer was admitted to the hospital for the treatment of a hepatic mass that was incidentally detected during evaluation of AoV cancer. Microscopically, the hepatic tumor was composed of solidly packed small, round and uniform undifferentiated cells, which resembled that of a small-blue-round-cell tumor. The immunophenotype of neoplastic cells (C-KIT+/EpCAM+/E-cadherin+/keratin 7−/keratin 19−/α-fetoprotein−/albumin−) supported primitive stem cell features with no hepatic or biliary phenotypes. Polymerase chain reaction and direct DNA sequencing revealed no C-KIT mutations. It is suggested that this tumor may have originated from transformed C-KIT+/EpCAM+/E-cadherin+ cells, which are more primitive and undifferentiated than bipotential hepatic progenitor cells.

Establishment of Hertwig's epithelial root sheath/epithelial rests of Malassez cell line from human periodontium.

Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-β1. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth.

The effects of histone deacetylase inhibitors on glioblastoma-derived stem cells.

Glioblastoma multiforme (GBM) is the most malignant brain tumor with limited effective treatment options. Cancer stem cells (CSCs), a subpopulation of cancer cells with stem cell properties found in GBMs, have been shown to be extremely resistant to radiation and chemotherapeutic agents and have the ability to readily reform tumors. Therefore, the development of therapeutic agents targeting CSCs is extremely important. In this study, we isolated glioblastoma-derived stem cells (GDSCs) from GBM tissue removed from patients during surgery and analyzed their gene expression using quantitative real-time PCR and immunocytochemistry. We examined the effects of histone deacetylase inhibitors trichostatin A (TSA) and valproic acid (VPA) on the proliferation and gene expression profiles of GDSCs. The GDSCs expressed significantly higher levels of both neural and embryonic stem cell markers compared to GBM cells expanded in conventional monolayer cultures. Treatment of GDSCs with histone deacetylase inhibitors, TSA and VPA, significantly reduced proliferation rates of the cells and expression of the stem cell markers, indicating differentiation of the cells. Since differentiation into GBM makes them susceptible to the conventional cancer treatments, we posit that use of histone deacetylase inhibitors may increase efficacy of the conventional cancer treatments for eliminating GDSCs.

Cripto-1 as a novel therapeutic target for triple negative breast cancer.

Triple-negative breast cancer (TNBC) presents the poorest prognosis among the breast cancer subtypes and no current standard therapy. Here, we performed an in-depth molecular analysis of a mouse model that establishes spontaneous lung metastasis from JygMC(A) cells. These primary tumors resembled the triple-negative breast cancer (TNBC) both phenotypically and molecularly. Morphologically, primary tumors presented both epithelial and spindle-like cells but displayed only adenocarcinoma-like features in lung parenchyma. The use of laser-capture microdissection combined with Nanostring mRNA and microRNA analysis revealed overexpression of either epithelial and miRNA-200 family or mesenchymal markers in adenocarcinoma and mesenchymal regions, respectively. Cripto-1, an embryonic stem cell marker, was present in spindle-like areas and its promoter showed activity in primary tumors. Cripto-1 knockout by the CRISPR-Cas9 system inhibited tumor growth and pulmonary metastasis. Our findings show characterization of a novel mouse model that mimics the TNBC and reveal Cripto-1 as a TNBC target hence may offer alternative treatment strategies for TNBC.

Zscan4 interacts directly with human Rap1 in cancer cells regardless of telomerase status

Telomeres are repetitive sequences at the ends of chromosomes protected by DNA binding proteins of the shelterin complex that form capping structures. Through the interaction of shelterin complex-associated proteins, telomere length maintenance is regulated. Recently, the newly identified embryonic stem cell marker, Zinc finger and SCAN domain-containing 4 gene (Zscan4), was shown to be a telomere-associated protein, co-localizing to the shelterin complex. Furthermore, it was shown to play an essential role in genomic stability by regulating telomere elongation. Although it is known that Zscan4 regulates TRF2, POT1b, and Rap1 expression in embryonic stem cells, the relationship and the exact mechanism of action for ZSscan4-mediated telomere maintenance in cancer cells is unknown. In this study, we investigated Zscan4 expression and interactions with Rap1 in telomerase positive (HeLa, MCF7) and ALT pathway (SaOS2, U2OS) cancer cells. Through western, pulldown, siRNA, and overexpression assays we demonstrate, for the first time, that Zscan4 directly associates with Rap1 (physical association protein). Furthermore, by generating truncated versions of Zscan4, we identified its zinc finger domain as the Rap1 binding site. Using bimolecular fluorescence complementation, we further validate this functional interaction in human cancer cells. Our results indicate that Zscan4 functions as a mediator of telomere length through its direct interaction with Rap1, possibly regulating shelterin complex-controlled telomere elongation in both telomerase positive and alternative lengthening of telomere pathways. This direct interaction between Zscan4 and Rap1 may explain how Zscan4 rapidly increases telomere length, yielding important information about the role of these proteins in telomere biology.

The role of H1 linker histone subtypes in preserving the fidelity of elaboration of mesendodermal and neuroectodermal lineages during embryonic development.

H1 linker histone proteins are essential for the structural and functional integrity of chromatin and for the fidelity of additional epigenetic modifications. Deletion of H1c, H1d and H1e in mice leads to embryonic lethality by mid-gestation with a broad spectrum of developmental alterations. To elucidate the cellular and molecular mechanisms underlying H1 linker histone developmental functions, we analyzed embryonic stem cells (ESCs) depleted of H1c, H1d and H1e subtypes (H1-KO ESCs) by utilizing established ESC differentiation paradigms. Our study revealed that although H1-KO ESCs continued to express core pluripotency genes and the embryonic stem cell markers, alkaline phosphatase and SSEA1, they exhibited enhanced cell death during embryoid body formation and during specification of mesendoderm and neuroectoderm. In addition, we demonstrated deregulation in the developmental programs of cardiomyocyte, hepatic and pancreatic lineage elaboration. Moreover, ectopic neurogenesis and cardiomyogenesis occurred during endoderm-derived pancreatic but not hepatic differentiation. Furthermore, neural differentiation paradigms revealed selective impairments in the specification and maturation of glutamatergic and dopaminergic neurons with accelerated maturation of glial lineages. These impairments were associated with deregulation in the expression profiles of pro-neural genes in dorsal and ventral forebrain-derived neural stem cell species. Taken together, these experimental observations suggest that H1 linker histone proteins are critical for the specification, maturation and fidelity of organ-specific cellular lineages derived from the three cardinal germ layers.

Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in embryonic stem cells.

Krüppel-like factors (Klf) 4 and 5 are two closely related members of the Klf family, known to play key roles in cell cycle regulation, somatic cell reprogramming and pluripotency. Here we focus on the functional divergence between Klf4 and Klf5 in the inhibition of mouse embryonic stem (ES) cell differentiation. Using microarrays and chromatin immunoprecipitation coupled to ultra-high-throughput DNA sequencing, we show that Klf4 negatively regulates the expression of endodermal markers in the undifferentiated ES cells, including transcription factors involved in the commitment of pluripotent stem cells to endoderm differentiation. Knockdown of Klf4 enhances differentiation towards visceral and definitive endoderm. In contrast, Klf5 negatively regulates the expression of mesodermal markers, some of which control commitment to the mesoderm lineage, and knockdown of Klf5 specifically enhances differentiation towards mesoderm. We conclude that Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in murine ES cells.

Role of tumor suppressor genes in the cancer-associated reprogramming of human induced pluripotent stem cells.

Because of their pluripotent characteristics, human induced pluripotent stem cells (iPSCs) possess great potential for therapeutic application and for the study of degenerative disorders. These cells are generated from normal somatic cells, multipotent stem cells, or cancer cells. They express embryonic stem cell markers, such as OCT4, SOX2, NANOG, SSEA-3, SSEA-4, and REX1, and can differentiate into all adult tissue types, both in vitro and in vivo. However, some of the pluripotency-promoting factors have been implicated in tumorigenesis. Here, we describe the merits of tumor suppresser genes as reprogramming factors for the generation of iPSCs without tumorigenic activity. The initial step of reprogramming is induction of the exogenous pluripotent factors to generate the oxidative stress that leads to senescence by DNA damage and metabolic stresses, thus inducing the expression of tumor suppressor genes such as p21CIP1 and p16INK4a through the activation of p53 to be the pre-induced pluripotent stem cells (pre-iPSCs). The later stage includes overcoming the barrier of reprogramming-induced senescence or cell-cycle arrest by shutting off the function of these tumor suppressor genes, followed by the induction of endogenous stemness genes for the full commitment of iPSCs (full-iPSCs). Thus, the reactive oxygen species (ROS) produced by oxidative stress might be critical for the induction of endogenous reprogramming-factor genes via epigenetic changes or antioxidant reactions. We also discuss the critical role of tumor suppressor genes in the evaluation of the tumorigenicity of human cancer cell-derived pluripotent stem cells, and describe how to overcome their tumorigenic properties for application in stem cell therapy in the field of regenerative medicine.

The endometrium of cycling cows contains populations of putative mesenchymal progenitor cells.

Endometrial stem cells have been identified in humans, mice and pigs. This study was designed to determine whether the uterine endometrium of cycling cows contains such cells, to identify markers of stemness and ultimately to isolate putative stem/progenitor cell and evaluate their capability to differentiate into mesodermal derivatives. Uteri from healthy cows in the early (days 1-5) and late luteal phases (days 13-18) of the oestrous cycle were collected. Total RNA and proteins were isolated and searched for gene markers of embryonic (OCT4, NANOG, SOX2) and mesenchymal (CD44, STAT3, CD-117) stem cells and for protein markers (Oct4, Sox2, Cd44) in Western blots or immunostaining of paraffin-embedded tissue. Primary cell cultures were isolated; characterized in terms of morphology, colony formation and gene/protein expression; and induced osteogenic and chondrogenic differentiation. We identified expression of embryonic (OCT4 and SOX2, but not NANOG) and mesenchymal (STAT3, CD44 and c-KIT) gene markers in the endometrium of cycling cows and the encoded proteins (Oct4, Sox2 and Cd44) in both stages of the oestrous cycle. Derived cell lines displayed essentially the same gene expression pattern; however, at the protein level, Oct4 was not detected. No clear influence of the stage of the oestrous cycle was found. Cell lines from late luteal phase displayed osteogenic and chondrogenic differentiation potential upon chemical stimulation. In this research, we demonstrated the presence of mesenchymal progenitor cell populations of apparently mesenchymal origin in the endometrium of cycling cows, in both the early and late phases of the oestrous cycle. The cells isolated from the late luteal phase were more acquiescent to differentiate into mesodermal derivatives than cells in the early luteal phase. Our findings might have implications for the understanding of uterine stem cell biology in cows and other farm animal species.

Characterization of common marmoset dysgerminoma-like tumor induced by the lentiviral expression of reprogramming factors.

Recent generation of induced pluripotent stem (iPSCs) has made a significant impact on the field of human regenerative medicine. Prior to the clinical application of iPSCs, testing of their safety and usefulness must be carried out using reliable animal models of various diseases. In order to generate iPSCs from common marmoset (CM; Callithrix jacchus), one of the most useful experimental animals, we have lentivirally transduced reprogramming factors, including POU5F1 (also known as OCT3/4), SOX2, KLF4, and c-MYC into CM fibroblasts. The cells formed round colonies expressing embryonic stem cell markers, however, they showed an abnormal karyotype denoted as 46, X, del(4q), +mar, and formed human dysgerminoma-like tumors in SCID mice, indicating that the transduction of reprogramming factors caused unexpected tumorigenesis of CM cells. Moreover, CM dysgerminoma-like tumors were highly sensitive to DNA-damaging agents, irradiation, and fibroblast growth factor receptor inhibitor, and their growth was dependent on c-MYC expression. These results indicate that DNA-damaging agents, irradiation, fibroblast growth factor receptor inhibitor, and c-MYC-targeted therapies might represent effective treatment strategies for unexpected tumors in patients receiving iPSC-based therapy.