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At least 19 recordsLinked to original sources
PubMed2001

Feeder-free growth of undifferentiated human embryonic stem cells.

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.

Cell Culture Techniques↗
PubMed2026

Establishment and longitudinal characterisation of a feeder-free embryonic stem-like cell line (ATES1) derived from blastomeres of the climbing perch, Anabas testudineus.

Embryonic stem (ES) cell-based platforms in non-model teleosts remain scarce, which may constrain in vitro studies in functional genomics and developmental biology in aquaculture-relevant species. Here, we report the derivation and characterisation of ATES1, an ES-like cell line derived from Anabas testudineus, a commercially important freshwater fish. ATES1 has been maintained for over 250 passages (> 1500 days) under feeder-free conditions in Leibovitz-15 medium supplemented with foetal bovine serum, fish serum, embryo extract, and human basic fibroblast growth factor (hbFGF). Proliferation was optimal at 28 °C, with both fish serum and hbFGF significantly enhancing cell growth. The cell line exhibited multiple characteristics associated with ES-like cells in vitro during early passages (approximately up to passages 25-35), including ES cell-like morphology, high self-renewal capacity (5-bromo-2'-deoxyuridine incorporation > 90% and a doubling time of 39.9 h), alkaline phosphatase activity, responsiveness to differentiation induction conditions, and expression of stemness-associated genes (sox2, klf4, sall4, nanog, myc). Immunocytochemistry confirmed Sox2 protein expression. However, real-time PCR revealed a significant decline in klf4 and sall4 expression, along with a lack of responsiveness to differentiation cues, increased heterogeneity, and the emergence of chromosomal abnormalities, suggesting compromised maintenance of ES-like properties during extended passaging. Despite these limitations, ATES1 remained continuously proliferative under feeder-free conditions and supported moderate to high (~ 45.8 ± 4.26%) non-viral transgene delivery efficiency via lipofection, suggesting its potential utility as a genetically manipulable in vitro system for future cellular and biotechnological applications in Anabas testudineus.

Animals↗
PubMed2004

Highly efficient mRNA-based gene transfer in feeder-free cultured H9 human embryonic stem cells.

Several protocols have been described for virus-based gene transfer in human embryonic stem (hES) cells, while efficient non-viral methods are currently non-existing. In this study, we investigated the efficiency of mRNA-based gene transfer in feeder-free cultured H9 hES cells, based on electroporation of in vitro transcribed mRNA encoding the enhanced green fluorescent protein (EGFP). Optimisation of culture and electroporation conditions for feeder-free cultured H9 hES cells resulted a highly pure, transgene-expressing (90% positive cells) H9 hES cell population.

Cell Survival↗
PubMed2003

Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells.

Embryonic stem (ES) cells can differentiate into many cell types and are expected to be useful for tissue engineering. Recent reports have shown that ES cells can differentiate into insulin-producing cells in response to the transient expression of the pdx-1 gene, after the removal of feeder cells. To investigate the lineage of insulin-producing cells and their in vitro differentiation, we introduced the betageo gene, encoding a beta-galactosidase-neomycin phosphotransferase fusion protein under the control of the mouse insulin 2 promoter, into ES cells that had been adapted to feeder-free culture, and analyzed insulin gene expression during their in vitro differentiation. We also examined the expression of transcription factors that are related to the differentiation of the pancreas. X-gal staining analysis revealed beta-galactosidase-positive cells on the surface and in the center of the embryoid body that proliferated during differentiation. Glucose-responsive insulin-producing cells, derived from our feeder-free ES cells, expressed insulin 2, pdx-1, Pax4, and Isl1 and also the glucagon, somatostatin, and PP genes. Moreover, the genes encoding p48, amylase, and carboxypeptidase A were also expressed. These results suggest that ES cells can differentiate not only into endocrine cells but also into exocrine cells of the pancreas, without the initiation of pdx-1 expression.

Animals↗
PubMed2004

Long-term culture of human embryonic stem cells in feeder-free conditions.

We have demonstrated previously that human embryonic stem (hES) cells possess a characteristic morphologic, antigenic, and molecular profile that can be used to assess the state of ES cells (Carpenter et al., [2004] Dev Dyn 229:243-258). In this manuscript, we have examined the long-term stability of three hES cell lines in feeder-free culture. We demonstrate that the expression of antigens and transcription factors, telomerase activity, telomere length, and karyotype appear stable for all three hES cell lines after continuous culture for over 1 yr. All three lines retained pluripotent differentiation in vitro and in vivo. Although hES cell lines were remarkably stable over the period of analysis, a detailed quantitative analysis of antigen expression by flow cytometry and gene expression by microarray suggested that cell lines show subtle differences in the expression of small subsets of genes upon long-term culture.

Animals↗
PubMed2004

Properties of four human embryonic stem cell lines maintained in a feeder-free culture system.

Several laboratories have begun evaluating human ES (hES) cell lines; however, direct comparisons between different hES cell lines have not been performed. We have characterized the properties of four human cell lines maintained in feeder-free culture conditions. Quantitative assessment of surface markers, microarray analysis of gene expression patterns, expression of SOX-2, UTF-1, Rex-1, OCT3/4, CRIPTO, and telomerase activity demonstrated similar patterns in all hES cell lines examined. Undifferentiated hES cells do not respond to neurotransmitters such as acetylcholine, glutamate, and gamma-aminobutyric acid. In addition, the undifferentiated hES cells possess gap junctions. Although similarities in marker expression were observed, allotyping showed that all four lines have a distinct HLA profile, predicting differences in transplantation responses. These data provide the first detailed comparison of different hES cell lines and demonstrate remarkable similarities among lines maintained in identical culture conditions.

Antigens, CD↗
PubMed2004

Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001.

The number of human embryonic stem cell (hESC) lines available to federally funded U.S. researchers is currently limited. Thus, determining their basic characteristics and disseminating these lines is important. In this report, we recovered and expanded the earliest available cryopreserved stocks of the BG01, BG02, and BG03 hESC lines. These cultures exhibited multiple definitive characteristics of undifferentiated cells, including long-term self-renewal, expression of markers of pluripotency, maintenance of a normal karyotype, and differentiation to mesoderm, endoderm, and ectoderm. Each cell line exhibited a unique genotype and human leukocyte antigen (HLA) isotype, confirming that they were isolated independently. BG01, BG02, and BG03 maintained in feederfree conditions demonstrated self-renewal, maintenance of normal karyotype, and gene expression indicative of undifferentiated pluripotent stem cells. A survey of gene expression in BG02 cells using massively parallel signature sequencing generated a digital read-out of transcript abundance and showed that this line was similar to other hESC lines. BG01, BG02, and BG03 hESCs are therefore independent, undifferentiated, and pluripotent lines that can be maintained without accumulation of karyotypic abnormalities.

Alkaline Phosphatase↗
PubMed2004

Long-term culture of mouse male germline stem cells under serum-or feeder-free conditions.

Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to the next generation. These cells can be cultured for extended periods in the presence of serum and feeder cells. However, little is known about factors that regulate self-renewal division of spermatogonial stem cells. In this investigation we examined the possibility of establishing culture systems for spermatogonial stem cells that lack serum or a feeder cell layer. Spermatogonial stem cells could expand in serum-free conditions on mouse embryonic fibroblasts (MEFs), or were successfully cultivated without feeder cells on a laminin-coated plate. However, they could not expand when both serum and feeder cells were absent. Although the cells cultured on laminin differed phenotypically from those on feeder cells, they grew exponentially for at least 6 mo, and produced normal, fertile progeny following transplantation into infertile mouse testis. This culture system will provide a new opportunity for understanding the regulatory mechanism that governs spermatogonial stem cells.

Animals↗
PubMed1991

v-abl does not abolish IL-6 requirement by murine plasmacytoma cells.

Activation of c-myc by juxtaposition to an immunoglobulin locus and introduction of the v-abl oncogene act synergistically in generating a mouse plasmacytoma (PC). The question arose whether the effect of v-abl could be attributed to a deregulation of interleukin-6 (IL-6) production or responsiveness, in view of the fact that IL-6 exerts potent growth-stimulatory activity on PC cells. We studied the effect of IL-6 on the in vitro growth of primary PCs induced by pristane alone (TEPCs) or by pristane + A-MuLV (ABPCs). Five of 13 TEPCs and 3 of 7 ABPCs responded to IL-6. Macrophage supernatants prepared from both TEPCs and ABPCs had similar stimulatory effects on PC cells. From 30 primary PCs (including both TEPCs and ABPCs), we established 9 in vitro lines, 2 of which expressed v-abl. All were able to grow on macrophage feeder layers. Three types of behavior could be distinguished on the basis of growth in feeder-free cultures in the presence and absence of IL-6. Group I contained 4 IL-6-dependent lines. Group II contained 2 IL-6-independent lines (one v-abl expressor) that grew faster in the presence of IL-6. Group III consisted of 3 feeder-dependent lines (one v-abl expressor) that were not significantly stimulated by IL-6. These findings indicate that v-abl expression does not influence IL-6 dependence or responsiveness by itself. The supernatant of one line in group II was able to stimulate PC cells. All 6 lines of Groups I and II carried a typical (12;15) translocation, while all 3 lines in group III had a variant (6;15) or (15;16) translocation.

Abelson murine leukemia virus↗
PubMed2027

Lentiviral Transduction of Embryonic Stem Cells.

Lentiviral vectors provide an efficient and reliable method for stable gene knockdown in embryonic stem cells (ESCs) through RNA interference. Here, we describe a detailed protocol for lentiviral transduction of mouse ESCs using lentiviral shRNA expression vectors. The protocol encompasses lentiviral particle production in HEK-293T packaging cells, determination of viral titer, transduction of ESCs cultured under feeder-free conditions, and selection of stably transduced cells. Additionally, we describe methods for evaluating transduction efficiency using fluorescence microscopy and flow cytometry, as well as for assessing gene knockdown efficacy by quantitative real-time PCR (Q-RT-PCR). This protocol is suitable for functional genomic studies in pluripotent stem cells and can be adapted for other difficult-to-transfect cell types.

Lentivirus↗
PubMed2004

Differences between human and mouse embryonic stem cells.

We compared gene expression profiles of mouse and human ES cells by immunocytochemistry, RT-PCR, and membrane-based focused cDNA array analysis. Several markers that in concert could distinguish undifferentiated ES cells from their differentiated progeny were identified. These included known markers such as SSEA antigens, OCT3/4, SOX-2, REX-1 and TERT, as well as additional markers such as UTF-1, TRF1, TRF2, connexin43, and connexin45, FGFR-4, ABCG-2, and Glut-1. A set of negative markers that confirm the absence of differentiation was also developed. These include genes characteristic of trophoectoderm, markers of germ layers, and of more specialized progenitor cells. While the expression of many of the markers was similar in mouse and human cells, significant differences were found in the expression of vimentin, beta-III tubulin, alpha-fetoprotein, eomesodermin, HEB, ARNT, and FoxD3 as well as in the expression of the LIF receptor complex LIFR/IL6ST (gp130). Profound differences in cell cycle regulation, control of apoptosis, and cytokine expression were uncovered using focused microarrays. The profile of gene expression observed in H1 cells was similar to that of two other human ES cell lines tested (line I-6 and clonal line-H9.2) and to feeder-free subclones of H1, H7, and H9, indicating that the observed differences between human and mouse ES cells were species-specific rather than arising from differences in culture conditions.

Animals↗
PubMed2003

Neural cells without functional N-Methyl-D-Aspartate (NMDA) receptors contribute extensively to normal postnatal brain development in efficiently generated chimaeric NMDA R1 -/- <--> +/+ mice.

Embryonic stem (ES) cells have revolutionised our understanding of animal physiology. Analysis of chimaeric mice generated from these cells allows us to study the role of genes in development and function of the nervous system. The NMDA receptor, one of the two major ionotropic glutamate receptors, has been proposed to play fundamental roles in the survival, migration, differentiation, and activity-dependent maturation of neural cells. The NMDA receptor subunit 1 (NR1) gene is indispensable for receptor function, and knock-out mice die at birth, inhibiting the study of glutamate signalling in postnatal neurons. Homozygous NR1-/- ES cells were derived from matings of heterozygous mice under feeder-free conditions. Chimaeras were made by incorporating these ES cells into wild-type blastocysts and by the classical aggregation of morulae between wild-type and NR1-/- embryos. The resulting chimaeras survive and develop normally. NR1-/- neurons, identified by their lacZ label, were analysed and quantified in developing and adult brains with varying knock-out contributions in every single brain region. Specifically, postnatal ontogenesis of cerebellum and hippocampus was normal. Accordingly, in chimaeric mice, NMDA receptor-initiated signals are not required for the migration, differentiation, and survival of most types of neurons in the central nervous system, in a cell-autonomous way.

Animals↗
PubMed2004

Maintenance and self-renewal of long-term reconstituting hematopoietic stem cells supported by amniotic fluid.

The maintenance and self-renewal of hematopoietic stem cells (HSC) in culture is a central focus of hematopoietic stem cell research. In vivo, the balance between HSC differentiation, apoptosis, and self-renewal is regulated at the endosteal surface niche in the bone marrow (BM). In feeder-free cultures, the fate of HSC is affected by growth factors/interleukins and serum, which affect the balance between self-renewal, differentiation, and apoptosis and lead to the rapid loss of multipotent HSC. We report that substituting human amniotic fluid (AF) for serum in HSC cultures provides a growth milieu in which HSC differentiation and apoptosis are down-regulated and multipotent HSC are maintained. Murine BM cells were cultured in serum-free medium containing 25% amniotic fluid and stem cell factor (SCF) only, "AF/SCF" cultures. Compared with serum and multiple growth factor-containing medium, cells cultured for 4 weeks in AF/SCF medium displayed downregulation of differentiation markers while maintaining a high fraction of cells expressing Sca1 (51.8%) and c-kit (10.2%). Reconstitution of lethally irradiated C57BL/6 (Ly5.2) mice with cultured Ly5.1 BM cells resulted in high levels of (cultured) donor cells in primary (78 +/- 19.4% and 94.32 +/- 2.5%, 10(5) and 10(6) cells injected, respectively) and secondary (96.5%) recipients at 8 and 11 months post-transplantation. Hence, long-term repopulation with AF/SCF cultured BM cells was maintained. Addition to the cultures of 10% serum, interleukin (IL)-3, IL-6, granulocyte colony stimulating factor (G-CSF), or granulocyte-macrophage colony stimulating factor (GM-CSF), singly or in combination, resulted in rapid differentiation and apoptosis, leading to the total loss of HSC.

Amniotic Fluid↗
PubMed2004

Monitoring early differentiation events in human embryonic stem cells by massively parallel signature sequencing and expressed sequence tag scan.

To identify genes that may be involved in the process of human embryonic stem cell (hESC) differentiation, we profiled gene expression by expressed sequenced tag (EST) enumeration and massively parallel signature sequencing (MPSS) using RNA samples from feeder-free cultures of undifferentiated (passages 40-50) and differentiated (day 14) H1, H7, and H9 lines. MPSS and EST scan analysis showed good concordance and identified a large number of genes that changed rapidly as cultures transition from a pluripotent to a differentiated state. These included known and unknown ES cell-specific genes as well as a large number of known genes that were altered as cells differentiate. A subset of genes that were either up- or down-regulated were selected and their differential expression confirmed by a variety of independent methods, including comparison of expression after further differentiation, publicly available databases, and direct assessments by reverse transcriptase (RT)-PCR and immunocytochemistry. The analysis identified markers unique to the hESC and embryoid bodies (hEBs) stage as well as signaling pathways that likely regulate differentiation. The data generated can be used to monitor the state of hESC isolated by different laboratories using independent methods and maintained under differing culture conditions.

Cell Culture Techniques↗
PubMed2004

Specification of the retinal fate of mouse embryonic stem cells by ectopic expression of Rx/rax, a homeobox gene.

With the goal of generating retinal cells from mouse embryonic stem (ES) cells by exogenous gene transfer, we introduced the Rx/rax transcription factor, which is expressed in immature retinal cells, into feeder-free mouse ES cells (CCE). CCE cells expressing Rx/rax as well as enhanced green fluorescent protein (CCE-RX/E cells) proliferated and remained in the undifferentiated state in the presence of leukemia inhibitory factor, as did parental ES cells. We made use of mouse embryo retinal explant cultures to address the differentiation ability of grafted ES cells. Dissociated embryoid bodies were treated with retinoic acid for use as donor cells and cocultured with retina explants for 2 weeks. In contrast to the parental CCE cells, which could not migrate into host retinal cultures, CCE-RX/E cells migrated into the host retina and extended their process-like structures between the host retinal cells. Most of the grafted CCE-RX/E cells became located in the ganglion cell and inner plexiform layers and expressed ganglion and horizontal cell markers. Furthermore, these grafted cells had the electrophysiological properties expected of ganglion cells. Our data thus suggest that subpopulations of retinal neurons can be generated in retinal explant cultures from grafted mouse ES cells ectopically expressing the transcription factor Rx/rax.

Animals↗
PubMed2004

LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells.

Murine embryonic stem (mES) cells remain undifferentiated in the presence of leukemia inhibitory factor (LIF), and activation of signal transducer and activator of transcription 3 (STAT3) via LIF receptor (LIFR) signaling appears sufficient for maintenance of mES cell pluripotency. Anecdotal and contradictory accounts exist for the action of LIF in the culture of human embryonic stem cells, and the nature of LIF signaling and whether the LIF-STAT3 pathway is conserved in human embryonic stem cells (hESCs) has not been systematically explored. In this study, we show that the LIFRbeta and the signaling subunit gp130 are expressed in hESCs and that human LIF can induce STAT3 phosphorylation and nuclear translocation in hESCs. Nevertheless, despite the functional activation of the LIF-STAT3 signaling pathway, human LIF is unable to maintain the pluripotent state of hESCs. Feeder-free culture conditions that maintain hESCs in an undifferentiated state do not show activation of STAT3, suggesting that distinct signaling mechanisms govern the self-renewal of hESCs.

Active Transport, Cell Nucleus↗
PubMed2004

Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth.

Human embryonic stem cells (hESCs) have the potential to generate multiple cell types and hold promise for future therapeutic applications. Although undifferentiated hESCs can proliferate indefinitely, hESC derivatives significantly downregulate telomerase and have limited replication potential. In this study we examine whether the replicative lifespan of hESC derivatives can be extended by ectopic expression of human telomerase reverse transcriptase (hTERT), the catalytic component of the telomerase complex. To this end, we have derived HEF1 cells, a fibroblast-like cell type, differentiated from hESCs. Infection of HEF1 cells with a retrovirus expressing hTERT extends their replicative capacity, resulting in immortal human HEF1-hTERT cells. HEF1-hTERT cells can be used to produce conditioned medium (CM) capable of supporting hESC growth under feeder-free conditions. Cultures maintained in HEF1-CM show characteristics similar to mouse embryonic fibroblast CM control cultures, including morphology, surface marker and transcription factor expression, telomerase activity, differentiation, and karyotypic stability. In addition, HEF1-hTERT cells have the capacity to differentiate into cells of the osteogenic lineage. These results suggest that immortalized cell lines can be generated from hESCs and that cells derived from hESCs can be used to support their own growth, creating a genotypically homogeneous system for the culture of hESCs.

Adipocytes↗
PubMed2026

Generation of Aneuploid Human Induced Pluripotent Stem Cells from Primary Amniotic Fluid Cells via Episomal Plasmid Electroporation.

The generation of patient-specific induced pluripotent stem cells (iPSCs) from amniotic fluid cells (AFCs) carrying defined chromosomal aneuploidies provides a powerful platform for modeling genetic disorders. However, establishing a reliable and reproducible reprogramming pipeline for aneuploid AFCs remains technically challenging due to the intrinsic genomic instability and variable proliferative capacity of these cells. Here, we present a comprehensive, non-integrating method for generating aneuploid human iPSCs from primary AFCs using episomal plasmid electroporation. This protocol details the complete workflow, encompassing cell thawing and expansion with a gradual media adaptation strategy, optimized plasmid delivery via electroporation system, sequential post-electroporation culture with mesenchymal-to-epithelial transition (MET)-directed media changes, and mechanical colony picking based on defined morphological criteria. We further describe validation procedures, including immunofluorescence staining for core pluripotency markers, G-banding karyotype analysis to confirm aneuploid karyotype maintenance, and PCR-based episomal vector clearance verification. This feeder-free, integration-free protocol yields aneuploid iPSC lines suitable for disease modeling, drug screening, and studies of chromosome biology.

Humans↗