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.
Highlights
Hematopoiesis is a complex and highly ordered process in which hematopoietic stem cells (HSCs) give rise to mature blood cells
We recently demonstrated that cell seeding density and bioreactor speed in both Spinner Flask and Synthecon systems significantly alters efficiency of embryonic stem cells (ESCs) differentiation into Linckit+ Sca-1+ cells [14]
Lineage negative (Lin-) ckit+Sca-1+ cells were isolated from mouse bone marrow (BM) and fetal liver (FL) cells
Summary
Hematopoiesis is a complex and highly ordered process in which hematopoietic stem cells (HSCs) give rise to mature blood cells. Most long-term multi-lineage HSC activity resides within the lineage-negative, ckit-positive, and Sca-1 positive fraction (Lin-ckit+Sca-1+, LKS cells) of murine bone marrow and fetal liver [2,3,4]. These cells have been shown to reconstitute in vivo all hematopoietic cells in mice following irradiation and hemato-lymphoid lineage depletion [5,6,7]. The clinical relevance of these cells has been further confirmed in humans after autologous HSC-rescue blood formation in myeloablated recipients, providing sustained hematopoiesis [8]. The clinical applicability of these ES/iPS-derived cells depends critically on (a) efficient methods for differentiation and expansion and (b) whether these cells are genetically and functionally equivalent to their native, in vivo counterparts from bone marrow (BM) or fetal liver (FL)
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