title: Gene expression profile and functionality of ESC-derived Lin-ckit+Sca-1+ cells are distinct from Lin-ckit+Sca-1+ cells isolated from fetal liver or bone marrow.
abstract: In vitro bioreactor-based cultures are being extensively investigated for large-scale production of differentiated cells from embryonic stem cells (ESCs). However, it is unclear whether in vitro ESC-derived progenitors have similar gene expression profiles and functionalities as their in vivo counterparts. This is crucial in establishing the validity of ESC-derived cells as replacements for adult-isolated cells for clinical therapies. In this study, we compared the gene expression profiles of Lin-ckit+Sca-1+ (LKS) cells generated in vitro from mouse ESCs using either static or bioreactor-based cultures, with that of native LKS cells isolated from mouse fetal liver (FL) or bone marrow (BM). We found that in vitro-generated LKS cells were more similar to FL- than to BM LKS cells in gene expression. Further, when compared to cells derived from bioreactor cultures, static culture-derived LKS cells showed fewer differentially expressed genes relative to both in vivo LKS populations. Overall, the expression of hematopoietic genes was lower in ESC-derived LKS cells compared to cells from BM and FL, while the levels of non-hematopoietic genes were up-regulated. In order to determine if these molecular profiles correlated with functionality, we evaluated ESC-derived LKS cells for in vitro hematopoietic-differentiation and colony formation (CFU assay). Although static culture-generated cells failed to form any colonies, they did differentiate into CD11c+ and B220+ cells indicating some hematopoietic potential. In contrast, bioreactor-derived LKS cells, when differentiated under the same conditions failed to produce any B220+ or CD11c+ cells and did not form colonies, indicating that these cells are not hematopoietic progenitors. We conclude that in vitro culture conditions significantly affect the transcriptome and functionality of ESC-derived LKS cells and although in vitro differentiated LKS cells were lineage negative and expressed both ckit and Sca-1, these cells, especially those obtained from dynamic cultures, are significantly different from native cells of the same phenotype.
authors.affiliation: Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Fernandez
authors.forename: Krista M
authors.initials: KM
authors.lastname: Fridley
authors.forename: Dhivya
authors.initials: D
authors.lastname: Arasappan
authors.forename: Rosalind V
authors.initials: RV
authors.lastname: Ambler
authors.forename: Philip W
authors.initials: PW
authors.lastname: Tucker
authors.forename: Krishnendu
authors.initials: K
authors.lastname: Roy
mesh_terms: D000818:Animals; D000950:Antigens, Ly / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism*; D001853:Bone Marrow / Q000254:growth & development / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005333:Fetus / Q000166:cytology / Q000378:metabolism*; D020869:Gene Expression Profiling; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000950:Antigens, Ly; D015415:Biomarkers; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D012333:RNA, Messenger; D019009:Proto-Oncogene Proteins c-kit