Embryonic Stem Cell-like Cells Research Articles

Optimization of Culture Conditions to Support Long-Term Self-Renewal of Buffalo (Bubalus bubalis) Embryonic Stem Cell-Like Cells

A culture system capable of sustaining self-renewal of buffalo embryonic stem (ES) cell-like cells in an undifferentiated state over a long period of time was developed. Inner cell masses were seeded on KO-DMEM+15% KO-serum replacer on buffalo fetal fibroblast feeder layer. Supplementation of culture medium with 5 ng/mL FGF-2 and 1000 IU/mL mLIF gave the highest (p<0.05) rate of primary colony formation. The ES cell-like cells' colony survival rate and increase in colony size were highest (p<0.05) following supplementation with FGF-2 and LIF compared to other groups examined. FGF-2 supplementation affected the quantitative expression of NANOG, SOX-2, ACTIVIN A, BMP 4, and TGFβ1, but not OCT4 and GREMLIN. Supplementation with SU5402, an FGFR inhibitor (≥20 μM) increased (p<0.05) the percentage of colonies that differentiated. FGFR1-3 and ERK1, K-RAS, E-RAS, and SHP-2, key signaling intermediates of FGF signaling, were detected in ES cell-like cells. Under culture conditions described, three ES cell lines were derived that, to date, have been maintained for 135, 95, and 85 passages for over 27, 19, and 17 months, respectively, whereas under other conditions examined, ES cell-like cells did not survive beyond passage 10. The ES cell-like cells were regularly monitored for expression of pluripotency markers and their potency to form embryoid bodies.

Cloning and characterization of buffalo NANOG gene: alternative transcription start sites, splicing, and polyadenylation in embryonic stem cell-like cells.

NANOG is a critical homeodomain transcription factor responsible for maintaining embryonic stem cell (ESC) self-renewal and pluripotency. In the present study, we isolated, sequenced, and characterized the NANOG gene in buffalo ESC-like cells. Here, we demonstrated that NANOG mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and five different polyadenylation sites. The TSSs identified by 5'-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5'-RACE) were positioned at 182, 95, 35, and 17 nucleotides upstream relative to the translation initiation codon. 3'-RACE experiment revealed the presence of tandem polyadenylation signals, which leads to the expression of at least five different 3'-untranslated regions (269, 314, 560, 566, and 829 nucleotides). Expression analysis showed that these alternatively polyadenylated transcripts expressed differentially. Sequence analysis showed that the open reading frame of buffalo NANOG codes for a 300-amino-acid-long protein. Further, results showed that alternative splicing leads to the expression of two types of transcript variants encoded by four and five exons. In silico analysis of cloned 5'-flanking region (3366 nucleotides upstream of translation start codon) identified several putative transcription factors binding sites in addition to a TATA box and CAAT box at -30 and -139 bp (upstream to the distal most TSS), respectively, in the buffalo NANOG promoter.

Isolation and Characterization of Embryonic Stem Cell-Like Cells From in vitro Produced Goat (Capra hircus) Embryos

The aim of the present study was to isolate and characterize goat embryonic stem cell-like cells from in vitro produced goat embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 150 blastocysts and 35 hatched blastocysts whereas 100 morulae were used for blastomeres isolation mechanically. The ICM derived cells or blastomeres were cultured on a feeder layer. The primary colony formation was significantly higher (P < 0.01) for hatched blastocysts (77.14%) than early/expanded blastocysts (54%) or morula (14%). When ICMs were isolated mechanically the primary colony formation for hatched blastocysts (90%) as well as blastocysts (66%) were significantly more than when ICMs were isolated by enzymatic digestion (60% and 30%, respectively). The colonies were disaggregated either mechanically or by enzymatic digestion for further subculture. When mechanical method was followed, the colonies remained undifferentiated up to 15 passages and three ES cell-like cell lines were produced (gES-1, gES-2, and gES-3). However, enzymatic disaggregation resulted in differentiation. The undifferentiated cells showed stem cell like morphological features, normal karyotype, and expressed stem cell specific surface markers like alkaline phosphatase, TRA-1-61, TRA-1-81, and intracellular markers Oct4, Sox2, and Nanog. Following prolonged culture of the ES cell-like cells were differentiated into several types of cells including neuron like and epithelium-like cells. In conclusion, goat embryonic stem cell-like cells can be isolated from in vitro produced goat embryos and can be maintained for long periods in culture.

Embryonic stem cell-like cells established by culture of adult human ovarian cells

Embryonic stem cell-like cells established by culture of adult human ovarian cells

Generation of Viable Embryos and Embryonic Stem Cell-Like Cells from Cultured Primary Follicles in Mice1

Primary follicles retrieved from B6CBAF1 prepubertal mice were cultured in a stepwise manner in an alpha-minimum essential medium-based medium to generate viable embryos and embryonic stem cell (ESC)-like cells. A significant increase in follicle growth and oocyte maturation accompanied by increased secretion of 17beta-estradiol and progesterone was achieved by exposing primary follicles to 100 or 200 mIU of follicle-stimulating hormone (FSH) during culture. More oocytes developed into blastocysts following in vitro fertilization (IVF) or parthenogenetic activation after culture with 200 mIU of FSH during the entire culture period than with 100 mIU. Eleven ESC-like cell lines, consisting of four heterozygotic and seven homozygotic phenotypes, were established from 25 trials of primary follicle culture combined with IVF or parthenogenetic activation. In conclusion, primary follicles can potentially yield developmentally competent oocytes, which produce viable embryos and ESC-like cell lines following in vitro manipulation. We suggest a method to utilize immature follicles, which are most abundant in ovaries, to improve reproductive efficiency and for use in regenerative medicine.

Reprogrammed Pluripotent Stem Cells from Somatic Cells

Pluripotent stem cells, such as embryonic stem (ES) cells, can differentiate into all cell types. So, these cells can be a biological resource for regenerative medicine. However, ES cells known as standard pluripotent cells have problem to be used for cell therapy because of ethical issue of the origin and immune response on the graft. Hence, recently reprogrammed pluripotent cells have been suggested as an alternative source for regenerative medicine. Somatic cells can acquire the ES cell-like pluripotency by transferring somatic cell nuclei into oocytes, by cell fusion with pluripotent cells. Retroviral-mediated introduction of four factors, Oct4, Sox2, Klf4 and c-Myc can successfully reprogram somatic cells into ES cell-like pluripotent stem cells, known as induced pluripotent stem (iPS) cells. These cells closely resemble ES cells in gene expression pattern, cell biologic and phenotypic characteristics. However, to reach the eventual goal of clinical application, it is necessary to overcome the major drawbacks such as low reprogramming efficiency and genomic alterations due to viral integration. In this review, we discuss the current reprogramming techniques and mechanisms of nuclear reprogramming induced by transcription factor transduction.

Effect of Cytoplasmic Volume on Developmental Competence of Buffalo (Bubalus bubalis) Embryos Produced Through Hand-Made Cloning

This study examined the effects of cytoplasmic volume on the developmental competence of hand-made cloned buffalo embryos. Two different cell types, that is, buffalo fetal fibroblast (BFF) and buffalo embryonic stem (ES) cell-like cells were taken as donor cell and fused with one, two, or three demicytoplasts to generate embryos with decreased, normal (control), and increased cytoplasmic volume. Using BFF as a nuclear donor, the cleavage rate was similar in all the groups (p > 0.05), but the blastocysts rate was significantly lower (p < 0.05) for embryos generated with decreased cytoplasmic volume. Using ES cell-like cells, the cleavage and blastocyst rate with increased cytoplasmic volume was significantly higher (p < 0.05) compared that with reduced cytoplasmic volume. Blastocysts produced from embryos having increased cytoplasmic volume had significantly higher (p < 0.05) cell number than normal (control) embryos in both BFF and ES cell-like cells groups. Pregnancies were established in all the groups except for the embryos reconstructed with decreased cytoplasmic volume. The pregnancy rate was almost double for embryos reconstructed using increased cytoplasmic volume compared to that with the controls. Most of the pregnancies aborted in the first trimester and one live calf was delivered through Caesarean, which died 4 h after birth.

Production of Cloned and Transgenic Embryos Using Buffalo (Bubalus bubalis) Embryonic Stem Cell-Like Cells Isolated fromIn VitroFertilized and Cloned Blastocysts

Here, we report the isolation and characterization of embryonic stem (ES) cell-like cells from cloned blastocysts, generated using fibroblasts derived from an adult buffalo (BAF). These nuclear transfer embryonic stem cell-like cells (NT-ES) grew in well-defined and dome-shaped colonies. The expression pattern of pluripotency marker genes was similar in both NT-ES and in vitro fertilization (IVF) embryo-derived embryonic stem cell-like cells (F-ES). Upon spontaneous differentiation via embryoid body formation, cells of different morphology were observed, among which predominant were endodermal-like and epithelial-like cell types. The ES cell-like cells could be passaged only mechanically and did not form colonies when plated as single cell suspension at different concentrations. When F-ES cell-like, NT-ES cell-like, and BAF cells of same genotype were used for hand-made cloning (HMC), no significant difference (p > 0.05) was observed in cleavage and blastocyst rate. Following transfer of HMC embryos to synchronized recipients, pregnancies were established only with F-ES cell-like and BAF cell-derived embryos, and one live calf was born from F-ES cell-like cells. Further, when transfected NT-ES cell-like cells and BAF were used for HMC, no significant difference (p > 0.05) was observed between cleavage and blastocyst rate. In conclusion, here we report for the first time the derivation of ES cell-like cells from an adult buffalo, and its genetic modification. We also report the birth of a live cloned calf from buffalo ES cell-like cells.

The Art of Human Induced Pluripotent Stem Cells: The Past, the Present and the Future

In 2006, Yamanaka and Takahashi electrified the scientific community by discovering that mouse somatic cells can be converted into embryonic stem cell-like cells by retroviral transduction of four transcription factors: Oct4, Sox2, Klf4, and c-Myc (OSKM). The first generation of mouse induced pluripotent stem (iPS) cells was incompletely reprogrammed, and failed to contribute to germline transmission. Nearly one year later, three groups, including Yamanaka's, improved the reprogramming methodology and generated iPS cells that were in many respects, indistinguishable from ES cells, and also contributed to chimera formation and germline transmission. Shortly thereafter, the successful reprogramming of human somatic cells opened the gate for the development of patient-specific iPS cells for biomedical research and clinical application. Though human iPS cells resemble human ES cells in many aspects, the current iPS cell technologies showed several limitations for clinical usage. First, the efficiency of iPS cell generation is still low and the reprogramming process takes at least two weeks. Second, the virus-delivery of reprogramming factors introduces inconceivable risks of insertional mutagenesis in the genome. Third, given the various strategies for direct reprogramming, it remains difficult to assess the quality of iPS cells generated in each lab and for each patient. These issues should be addressed properly before any iPS cells could be translated into clinic. Here, we review recent progress in human iPS cell technologies, with a focus on the virus-free and integration-free iPS cell generation, which may lead towards the eventual goal of clinical applications. Keyword: Human induced pluripotent stem cells (hiPSCs), reprogramming strategy, hiPSC efficiency.

Embryonic stem cell-like cells established by culture of adult ovarian cells in mice

To suggest an alternative strategy for deriving histocompatible stems cells without undertaking genetic manipulation. Prospective approach using an animal model. Stem cell and bioevaluation laboratory, Seoul National University. F1 (C57BL6 X DBA2) and outbred (ICR) mice. Ovarian stroma cells of less than 40 mum in diameter were subcultured with fibroblast monolayer, and colony-forming cells were characterized. Stemness, genotype, and imprinted gene methylation. Two-lines of colony-forming cells were established, which expressed markers specific for embryonic stem cells (ESC) and formed embryoid bodies and teratomas. Complete matching of microsatellite markers with the cell donor strain confirmed their establishment from ovarian tissue, and identification of both homozygotic and heterozygotic chromosomes raised the possibility of their derivation from parthenogenetic oocytes. However, the use of cells smaller than mature oocytes for primary culture, the difference in imprinted gene methylation compared with parthenogenetic ESCs, and failure to establish the ESC-like cells by primary follicle culture collectively suggested the irrelevancy to gametes. Coculture of adult ovarian cells with somatic fibroblasts can yield colony-forming cells having ESC-like activity, which may provide an alternative for establishing autologous stem cells from adults that can be obtained without genetic manipulation.

Generation of Induced Pluripotent Stem Cells by Efficient Reprogramming of Adult Bone Marrow Cells

Reprogramming of somatic cells provides potential for the generation of specific cell types, which could be a key step in the study and treatment of human diseases. In vitro reprogramming of somatic cells into a pluripotent embryonic stem (ES) cell-like state has been reported by retroviral transduction of murine fibroblasts using four embryonic transcription factors or through cell fusion of somatic and pluripotent stem cells. Here we show that mouse adult bone marrow mononuclear cells (BM MNCs) are competent as donor cells and can be reprogrammed into pluripotent ES cell-like cells. We isolated BM MNCs and mouse embryonic fibroblasts (MEFs) from Oct4-GFP transgenic mice, fused them with ES cells, or infected them with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc. Fused BM MNCs formed more ES-like colonies than did MEFs. Infected BM MNCs gave rise to induced pluripotent stem (iPS) cells, although transduction efficiencies were not high. It was more efficient to pick up iPS colonies as compared with MEFs. BM-derived iPS (BM iPS) cells expressed ES cell markers, formed teratomas, and contributed to chimera mice with germ line development. Clonal analysis revealed that BM iPS clones had diversity, although some clones were found to be genetically identical with different phenotypes. Our findings imply that BM MNCs have potential advantages to generate iPS cells for the clinical application.

Culture Conditions and Enzymatic Passaging of Bovine ESC-Like Cells

The goals of the current study were to (1) improve culture conditions and (2) chemical passaging of bovine embryonic stem cell-like (bESC-like) cells. Specifically, the effects of human leukemia inhibitory factor (hLIF), two types of feeders, mouse embryonic fibroblast (MEF) and bovine embryonic fibroblast (BEF), as well as three different enzymatic treatments including Trypsin-EDTA, TrypLE, and Liberase Blendzymes 3 were investigated. The addition of hLIF at 1000 U/mL to the culture medium (41.2 and 36.9%), and the use of either MEF or BEF feeders (40.3 and 38.1%) had no significant effect on the ability of inner cell masses (ICMs) to form primary cell colonies compared to controls. All bESC-like cells were first dissociated mechanically for three passages followed by enzymatic dissociation. The ability to maintain ESC morphology to passage 10 was compared among the three enzymes above. More bESC-like cell lines survived beyond passage 10 when treated with TrypLE compared to Trypson-EDTA (28.8 and 12.6%; p < 0.05), and bESC-like cells differentiated quickly when treated with Liberase Blendzyme 3. The bESC-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as SSEA-1, AP, OCT-4, and Nanog. When removed from feeders, these bESC-like cells formed embryoid bodies (EBs) in a suspension culture. When EBs were cultured on tissue culture plates, they differentiated into various cell types. In summary, we were able to culture bESC-like cells more than 10 passages by enzymatic dissociation, which is important in gene targeting, maintenance, and banking of bESC lines.

The bioethics of stem cell research and therapy

Discussion of the bioethics of human stem cell research has transitioned from controversies over the source of human embryonic stem cells to concerns about the ethical use of stem cells in basic and clinical research. Key areas in this evolving ethical discourse include the derivation and use of other human embryonic stem cell-like stem cells that have the capacity to differentiate into all types of human tissue and the use of all types of stem cells in clinical research. Each of these issues is discussed as I summarize the past, present, and future bioethical issues in stem cell research.

Hybrid cells differentiate to hepatic lineage cells and repair oxidative damage

Hybrid cells derived from stem cells play an important role in organogenesis, tissue regeneration and cancer formation. However, the fate of hybrid cells and their range of function are poorly understood. Fusing stem cells and somatic cells induces somatic cell reprogramming, and the resulting hybrid cells are embryonic stem cell-like cells. Therefore, we hypothesize that fusion-induced hybrid cells may behave like ES cells in certain microenvironments. In this study, human hepatic cells were induced to apoptosis with H2O2, and then co-cultured with hybrid cells that had been derived from mouse ES cells and human hepatic cells using a transwell. After co-culturing, the degree of apoptosis was evaluated using Annexin-V/PI double-staining analysis, flow cytometry and Western-blot. We observed that H2O2-induced cell apoptosis was inhibited by co-culture. In addition, the activity of injury-related enzymes (GSH-Px, LDH and SOD) and the level of albumin release in the co-culture system trended toward the level of normal undamaged hepatic cells. The stably increased levels of secretion of ALB in the co-culture system also confirmed that co-culture with hybrid cells helped in recovery from injury. The fate of the hybrid cells was studied by analyzing their gene expression and protein expression profiles. The results of RT-PCR indicated that during co-culturing, like ES cells, hybrid cells differentiated into hepatic lineage cells. Hybrid cells transcripted genes from both parental cell genomes. Via immunocytochemical analysis, hepatic directional differentiation of the hybrid cells was also confirmed. After injecting the hybrid cells into the mouse liver, the GFP-labeled transplanted cells were distributed in the hepatic lobules and engrafted into the liver structure. This research expands the knowledge of fusion-related events and the possible function of hybrid cells. Moreover, it could indicate a new route of differentiation from pluripotent cells to tissue-specific cells via conditional co-culture.Electronic Supplementary MaterialSupplementary material is available for this article at 10.2478/s11658-010-0018-0 and is accessible for authorized users.

Embryonic stem cell-like cells derived from adult human testis

Given the significant drawbacks of using human embryonic stem (hES) cells for regenerative medicine, the search for alternative sources of multipotent cells is ongoing. Studies in mice have shown that multipotent ES-like cells can be derived from neonatal and adult testis. Here we report the derivation of ES-like cells from adult human testis. Testis material was donated for research by four men undergoing bilateral castration as part of prostate cancer treatment. Testicular cells were cultured using StemPro medium. Colonies that appeared sharp edged and compact were collected and subcultured under hES-specific conditions. Molecular characterization of these colonies was performed using RT-PCR and immunohistochemistry. (Epi)genetic stability was tested using bisulphite sequencing and karyotype analysis. Directed differentiation protocols in vitro were performed to investigate the potency of these cells and the cells were injected into immunocompromised mice to investigate their tumorigenicity. In testicular cell cultures from all four men, sharp-edged and compact colonies appeared between 3 and 8 weeks. Subcultured cells from these colonies showed alkaline phosphatase activity and expressed hES cell-specific genes (Pou5f1, Sox2, Cripto1, Dnmt3b), proteins and carbohydrate antigens (POU5F1, NANOG, SOX2 and TRA-1-60, TRA-1-81, SSEA4). These ES-like cells were able to differentiate in vitro into derivatives of all three germ layers including neural, epithelial, osteogenic, myogenic, adipocyte and pancreatic lineages. The pancreatic beta cells were able to produce insulin in response to glucose and osteogenic-differentiated cells showed deposition of phosphate and calcium, demonstrating their functional capacity. Although we observed small areas with differentiated cell types of human origin, we never observed extensive teratomas upon injection of testis-derived ES-like cells into immunocompromised mice. Multipotent cells can be established from adult human testis. Their easy accessibility and ethical acceptability as well as their non-tumorigenic and autogenic nature make these cells an attractive alternative to human ES cells for future stem cell therapies.

From boar permatogenic cells to embryonic stem cell-like cells

The ubiquity, structural variation, and functional diversity of iron-sulfur (Fe-S) clusters in biological environments has long fascinated scientists. In nature, Fe-S clusters form a variety of shapes and sizes, from the simple [Fe2S2] rhombic cluster to the complex [Fe8S7] fused di-cubane ‘P-cluster’, among others. Fe-S clusters perform critical tasks, including electron transfer, catalysis, sulfur donation, and sensing, in many essential biological processes, such as cellular respiration, gene expression, nitrogen fixation, DNA repair, cofactor biosynthesis, and more. To further understand Fe-S clusters, they have been synthesized outside of their protein environments with various monodentate, bidentate, and tridentate ligands. In addition to providing insight into their biological properties, synthetic small molecule models of Fe-S clusters have yielded oxidation states and structures beyond those observed in biology as well as led to the development of many small molecule catalysts. More recently, Fe-S clusters have been utilized in an expanding array of applications, including maquettes and artificial proteins, biomimetic materials, and therapeutic development. The high degree of utility of Fe-S clusters outside of their biological roles suggests exciting possibilities for future uses of these earth-abundant, redox-active, and structurally and functionally diverse clusters. This review provides an overview of Fe-S clusters in and beyond biology to highlight the growing versatile uses of these clusters. Our discussion is divided into sections that describe the structure–function relationship of Fe-S clusters in five research areas: biology, synthetic small molecules, maquettes and artificial proteins, biomimetic materials, and therapeutic development. We also tracked the number of publications in each research area by year to identify trends in Fe-S cluster research, and we conclude by describing the overarching connections between each of the research areas. Overall, this review summarizes the application areas of Fe-S cluster research and highlights connections between the areas based on the structure–function relationship of Fe-S clusters and their ligands.

Krüppel-like factor 4, a “pluripotency transcription factor” highly expressed in male postmeiotic germ cells, is dispensable for spermatogenesis in the mouse

Krüppel-like factor 4 (Klf4, GKLF) was originally characterized as a zinc finger transcription factor essential for terminal differentiation and cell lineage allocation of several cell types in the mouse. Mice lacking Klf4 die postnatally within hours due to impaired skin barrier function and subsequent dehydration. Recently, KLF4 was also used in cooperation with other transcription factors to reprogram differentiated cells to pluripotent embryonic stem cell-like cells. Moreover, involvement in oncogenesis was also ascribed to KLF4, which is aberrantly expressed in some types of tumors such as breast, gastric and colon cancer. We previously have shown that Klf4 is strongly expressed in postmeiotic germ cells of mouse and human testes suggesting a role for Klf4 also during spermiogenesis. In order to analyze its function we deleted Klf4 in germ cells using the Cre–loxP system. Homologous recombination of the Klf4 locus has been confirmed by genomic southern blotting and the absence of the protein in germ cells was demonstrated by Western blotting and immunofluorescence. Despite its important roles in several significant biological settings, deletion of Klf4 in germ cells did not impair spermiogenesis. Histologically, the mutant testes appeared normal and the mice were fertile. In order to identify genes that were regulated by KLF4 in male germ cells we performed microarray analyses using a whole genome array. We identified many genes exhibiting changed expression in mutants even including the telomerase reverse transcriptase mRNA, which is a stem cell marker. However, in summary, the lack of KLF4 alone does not prevent complete spermatogenesis.

Recreating Stem Cells: A Novel Entrance to the Fountain of Youth

Differentiated cells can be reprogrammed to form pluripotent embryonic stem cell-like cells by ectopic expression of four transcription factors. In this issue of Cell Stem Cell, Liu et al. (2009) demonstrate that a similar transition can be achieved by culturing retinoblastoma-deficient mouse embryonic fibroblasts in suspension.

Low immunogenicity of endothelial derivatives from rat embryonic stem cell-like cells

Embryonic stem cells (ESC) are suggested to be immune-privileged, but they carry the risk of uncontrolled expansion and malignancy. Upon differentiation they lose their tumor-forming capacity, but they become immunogenic by the expression of a normal set of MHC molecules. This immunogenicity might trigger rejection after application in regenerative therapies. In this study MHC expression of and immune responses to endothelial derivatives of rat embryonic stem cell-like cells (RESC) under inflammatory conditions were determined in comparison to primary rat aortic endothelial cells (ECs). Cellular as well as humoral allo-recognition was analyzed in vitro. In addition, immune reactions in vivo were assessed by allo-antibody production and determination of interferon-gamma (IFNgamma)-secreting allo-reactive T cells. RESC derivatives expressed low but significant levels of MHC class I, and no MHC class II. In response to IFNgamma MHC class I expression was enhanced, while class II transactivator induction failed completely in these cells; MHC class II expression remained consistently absent. Functionally, the RESC derivatives showed a reduced allo-stimulatory capacity, protection against humoral allo-recognition in vitro and a slightly diminished susceptibility to cytotoxic T cell lysis. Furthermore, in vivo experiments demonstrated that these cells do not trigger host immune reactions, characterized by no allo-antibody production and no induction of allo-reactive memory T cells. Our results show that endothelial derivatives of RESC have a distinctive reduced immunogenic potency even under inflammatory conditions.

Three-Dimensional Synthetic Niche Components to Control Germ Cell Proliferation

Spermatogonial stem cells (SSCs) are increasingly studied for potential use in tissue regeneration due to their ability to dedifferentiate into embryonic stem cell-like cells. For their successful therapeutic use, these cells must first be expanded in vitro using an appropriate culture system. We hypothesized that a hydrogel with proper biochemical and biomechanical properties may mimic the composition and structure of the native basement membrane onto which SSCs reside, thus allowing us to control SSC proliferation. This hypothesis was examined in two-dimensional (2D) and three-dimensional (3D) cultures using hydrogels formed from calcium cross-linked alginate molecules conjugated with synthetic oligopeptides containing the Arg-Gly-Asp sequence (RGD peptides). The RGD peptide density (N(RGD)) in gel matrices was controlled by mixing alginate molecules modified with RGD peptides and unmodified alginate molecules at varied ratios. The mechanical stiffness was controlled with the cross-linking density of gel matrices. Interestingly, the RGD peptide density modulated cell proliferation in both 2D and 3D cultures as well as the number and size of SSC colonies formed in 3D cultures. In contrast, cell proliferation was minimally influenced by mechanical stiffness in 2D cultures. Overall, the results of this study elucidate an important factor regulating SSC proliferation and also present a bioactive hydrogel that can be used as a 3D synthetic basement membrane. In addition, the results of this study will be broadly useful in controlling the proliferation of various stem cells.