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A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: comparative expression profiles of mRNAs, microRNAs and proteins among different feeders and conditioned media.

ABSTRACT: BACKGROUND: Human embryonic stem (hES) cell lines were derived from the inner cell mass of human blastocysts, and were cultured on mouse embryonic fibroblast (MEF) feeder to maintain undifferentiated growth, extensive renewal capacity, and pluripotency. The hES-T3 cell line with normal female karyotype was previously used to differentiate into autogeneic fibroblast-like cells (T3HDF) as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF) for 14 passages. RESULTS: A feeder-free culture on Matrigel in hES medium conditioned by the autogeneic feeder cells (T3HDF) was established to maintain the undifferentiated growth of hES-T3 cells (T3/CMHDF) for 8 passages in this investigation. The gene expression profiles of mRNAs, microRNAs and proteins between the undifferentiated T3/HDF and T3/CMHDF cells were shown to be very similar, and their expression profiles were also found to be similar to those of T3/MEF and T3/CMMEF cells grown on MEF feeder and feeder-free Matrigel in MEF-conditioned medium, respectively. The undifferentiated state of T3/HDF and T3/CMHDF as well as T3/MEF and T3/CMMEF cells was evidenced by the very high expression levels of "stemness" genes and low expression levels of differentiation markers of ectoderm, mesoderm and endoderm in addition to the strong staining of OCT4 and NANOG. CONCLUSION: The T3HDF feeder and T3HDF-conditioned medium were able to support the undifferentiated growth of hES cells, and they would be useful for drug development and toxicity testing in addition to the reduced risks of xenogeneic pathogens when used for medical applications such as cell therapies.

SUBMITTER: Tsai ZY

PROVIDER: S-EPMC2958897 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: comparative expression profiles of mRNAs, microRNAs and proteins among different feeders and conditioned media.

Tsai Zong-Yun ZY Singh Sher S Yu Sung-Liang SL Chou Chi-Hsien CH Li Steven Shoei-Lung SS

BMC cell biology 20101012

<h4>Background</h4>Human embryonic stem (hES) cell lines were derived from the inner cell mass of human blastocysts, and were cultured on mouse embryonic fibroblast (MEF) feeder to maintain undifferentiated growth, extensive renewal capacity, and pluripotency. The hES-T3 cell line with normal female karyotype was previously used to differentiate into autogeneic fibroblast-like cells (T3HDF) as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF) for 14 passages.<h4>Results</h4> ...[more]

PMID: 20937144

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Project description:Background: The hES-T3 cell line with normal female karyotype was used to differentiate into autogeneic fibroblast-like cells (T3HDF) as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF) for 14 passages. A feeder-free culture on Matrigel in hES medium conditioned by these autogeneic feeder cells (T3HDF) was established to maintain the undifferentiated growth of hES-T3 cells (T3/CMHDF) for 8 passages. Results: The gene expression profiles of mRNAs, microRNAs and proteins between the undifferentiated T3/HDF and T3/CMHDF cells were shown to be very similar, and their expression profiles were also found to be similar to those of T3/MEF and T3/CMMEF cells grown on MEF feeder and feeder-free Matrigel in MEF-conditioned medium, respectively. The abundantly expressed genes of T3/HDF, T3/CMHDF, T3/MEF and T3/CMMEF cells were found to play prominent roles in signaling pathways and GO process networks. The miR-302/367 cluster and miR-371/372/373 cluster were extremely abundantly expressed in undifferentiated T3/HDF and T3/CMHDF as well as T3/MEF andT3/CMMEF cells. The undifferentiated state of T3/HDF and T3/CMHDF cells was evidenced by the very high expression levels of ¡§stemness¡¨ genes and low expression levels of differentiation markers of ectoderm, mesoderm and endoderm in addition to the strong staining of OCT4 and NANOG. Conclusion: The T3HDF feeder and T3HDF-conditioned medium were able to support the undifferentiated growth of hES cells, and they would be useful for drug development and toxicity testing in addition to the reduced risks of xenogeneic pathogens when used for medical applications such as cell therapies. Keywords: genetic modification In this investigation, both miRNA and mRNA expression profiles from human embryonic stem cells (hES-T3) grown on MEF feeder (T3_MEF), feeder-free Matrigel in MEF-conditioned medium (T3_CMMEF), T3HDF (hES-T3 differentiated fibroblast-like cells) feeder and feeder-free Matrigel in T3HDF-conditioned medium were quantitatively determined. Several target genes of T3BA and T3CM cells-specific miRNAs were identified. ***This submission represents the mRNA expression component of the study only***

Project description:BackgroundDespite a long history of investigation and sustained efforts in clinical testing, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration. Several barriers are impeding the clinical progression of MSC therapies, to the forefront of these is a lack of standardised manufacturing protocols which is further compounded by an absence of biologically meaningful characterisation and release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, among other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action.MethodsThis work analysed the influence of five widely used culture media formulations on the colony-forming potential, proliferation kinetics, trilineage differentiation potential and immunomodulatory potential of human bone marrow-derived MSCs (BM-MSCs). The surface marker expression profiles were also characterised using a high-content flow cytometry screening panel of 243 markers.ResultsSignificant differences in the biological attributes of BM-MSCs including clonogenicity, proliferation, differentiation propensity and immunomodulatory capacity were revealed in response to the composition of the culture medium. Despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs: CD73, CD90 and CD105. Immunophenotypic profiling revealed that the culture medium also had a significant influence on the surface proteome, with one-third of tested markers exhibiting variable expression profiles. Principal component analysis demonstrated that BM-MSCs isolated and expanded in a proprietary xeno- and serum-free medium displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate and foetal bovine serum-containing media.ConclusionsThese data suggest that media composition has a highly significant impact on the biological attributes of MSCs, but standard surface marker tests conceal these differences. The results indicate a need for (1) standardised approaches to manufacturing, with an essential focus on defined media and (2) new biologically relevant tests for MSC characterisation and product release.

Dataset Information

A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: comparative expression profiles of mRNAs, microRNAs and proteins among different feeders and conditioned media.

Publications

A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: comparative expression profiles of mRNAs, microRNAs and proteins among different feeders and conditioned media.

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