Unknown,Transcriptomics,Genomics,Proteomics

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Homogeneous monocytes and macrophages from hES cells following coculture-free differentiation in M-CSF and IL-3.

ABSTRACT: In the present study we aimed at producing homogeneous populations of monocytes and macrophages from human embryonic stem (hES) cells. Human embryonic stem cell lines KCL001, KCL002, and HUES-2 were differentiated into monocytes by coculture-free differentiation with two growth factors using a three-step method. The method involved embryoid body (EB) formation in hES media, directed differentiation with macrophage colony-stimulating factor and interleukin (IL)-3, and harvest of nonadherent monocytes from the culture supernatants. Transcriptome analysis of the esMC and the esMDM revealed a distinct myeloid signature that correlated with primary adult blood-derived monocytes and spleen tissue samples but not with other tissue samples tested. In conclusion we have developed a simple and efficient method for producing homogeneous populations of monocytes and macrophages from hES cells. esMCs have a myeloid signature and can differentiate into functional macrophages. The method should prove useful in answering experimental questions regarding monocyte and macrophage development and biology. Triplicates of monocytes and macrophages derived from human embryonic stem cells were compared to a triplicate of freshly isolated human blood monocytes.

ORGANISM(S): Homo sapiens

SUBMITTER: Fernando Martinez Estrada

PROVIDER: E-GEOD-15791 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Homogeneous monocytes and macrophages from human embryonic stem cells following coculture-free differentiation in M-CSF and IL-3.

Karlsson Karl R KR Cowley Sally S Martinez Fernando O FO Shaw Michael M Minger Stephen L SL James William W

Experimental hematology 20080611 9

<h4>Objective</h4>To develop a simple and efficient method for producing homogeneous populations of monocytes and macrophages from human embryonic stem cells (hES).<h4>Materials and methods</h4>Human embryonic stem cell lines KCL001, KCL002, and HUES-2 were differentiated into monocytes by coculture-free differentiation with two growth factors using a three-step method. The method involved embryoid body (EB) formation in hES media, directed differentiation with macrophage colony-stimulating fact ...[more]

PMID: 18550257

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Project description:Human embryonic stem (hES) cells have the capacities to propagate for extended periods and to differentiate into cell types from all three germ layers both in vitro and in vivo. These characteristics of self-renewal and pluripotency enable hES cells having the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics studies. The hES-T3 cells with normal female karyotype cultured on either mouse embryonic fibroblasts (MEF) in hES medium (containing 4 ng/ml bFGF) (T3MF) or feeder-free Matrigel in MEF-conditioned medium (supplemented with additional 4 ng/ml bFGF) (T3CM) were found to express very similar profiles of mRNAs and microRNAs, indicating that the unlimited self-renewal and pluripotency of hES cells can be maintained by continuing culture on these two conditions. However, the expression profiles, especially microRNAs, of the hES-T3 cells cultured on Matrigel in hES medium supplemented with 4 ng/ml bFGF and 5 ng/ml activin A (T3BA) were found to be different from those of T3MF and T3CM cells. In T3BA cells, four hES cell-specific microRNAs miR-372, miR-302d, miR-367 and miR-200c, as well as three other microRNAs miR-199a, miR-19a and miR-217, were found to be up-regulated, whereas five miRNAs miR-19b, miR-221, miR-222, let-7b and let-7c were down-regulated by activin A. Thirteen abundantly differentially expressed mRNAs, including NR4A2, ERBB4, CXCR4, PCDH9, TMEFF2, CD24 and COX6A1 genes, targeted by seven over-expressed miRNAs were identified by inverse expression levels of these seven microRNAs to their target mRNAs in T3BA and T3CM cells. The NR4A2, ERBB4 and CXCR4 target genes were further found to be regulated by EGF and/or TNF. The 50 abundantly differentially expressed genes targeted by five under-expressed miRNAs were also identified. The abundantly expressed mRNAs in T3BA and T3CM cells were also analyzed for the network and signaling pathways, and roles of activin A in cell proliferation and differentiation were found. These findings will help elucidate the complex signaling network which maintains the self-renewal and pluripotency of hES cells. In this investigation, both miRNA and mRNA expression profiles from human embryonic stem cells grown on MEF feeder (T3MF), feeder-free Matrigel in MEF-conditioned medium (T3CM) and in hES medium (containing 4 ng/ml bFGF) supplemented with 5 ng/ml activin A (T3BA) 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: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***

Dataset Information

Homogeneous monocytes and macrophages from hES cells following coculture-free differentiation in M-CSF and IL-3.

Publications

Homogeneous monocytes and macrophages from human embryonic stem cells following coculture-free differentiation in M-CSF and IL-3.

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