Functionality of various LKS cells.

<p>Hematopoietic differentiation of <i>in-vitro and in-vivo</i> derived Lin-ckit+Sca-1+ cells. <b>A)</b> Lin-ckit+Sca-1+ <i>in-vitro</i> derived ESCs from Static and Dynamic (Synthecon) culture conditions, as well as <b>B)</b><i>in-vivo</i> isolated LKS cells from Bone marrow (BM) were cultured for 7 days in the presence of GM-CSF, IL-4 and IL3. Approximately 10.3% of Static-derived cells (gated on FSC-A and SSC-A) are CD11c+ (Dendritic-like cells) and about 11.5% express B220+ (B cell-like progenitors). No significant expression of CD11c or B220 in Synthecon-derived cells is observed following differentiation. As expected, Lin-ckit+Sca-1+ BM cells also differentiate into CD11c and B220+ hematopoietic progenitors.</p

Global gene expression profile of BM- and FL-isolated as well as in vitro generated Lin-ckit+Sca-1+ progenitor cells.

<p><b>A)</b> Sample Hierarchical clustering represents the conditions that cluster together based on differentially expressed genes (DEG) across all samples (mean of gene expression values for each group, One way Anova, p<0.05). <b>B)</b> Gene Hierarchical clustering of 5-fold DEG across native (BM+FL),Static and Dynamic (Spinner+Synthecon) culture conditions. Gene Hierarchical clustering of 5-fold DEG up-regulated (<b>C</b>) or down-regulated (<b>D</b>) in Static and Dynamic (Spinner and Synthecon) <i>in vitro</i> culture conditions compared to native isolated LKS cells (BM+FL). The genes used in these heat maps are listed within Supplementary material.</p

Identification and isolation of ES cells and BM- and FL-derived Lin-ckit+Sca-1+ cells.

<p>(<b>A</b>) Lin-ckit+Sca-1+ cells were generated from ES cells differentiated under Static and Dynamic culture conditions (Synthecon and Spinner flask) for 7 days and (<b>B</b>) from BM and FL. Mouse embryonic stem cells were differentiated under Static and Dynamic culture conditions (Spinner flask and Synthecon) for 7 days. Cell suspensions from day7 EBs were stained with ckit and Sca-1 antibodies to isolate the Lin-ckit+Sca-1+ population. As explained in Materials and Methods, the Lineage negative cocktail was not included for sorting because we did not observe lineage positive cells at day 7 of differentiation for any ESC culture condition (left panel). Similarly, lineage negative cells from BM and FL cell suspensions were stained with ckit and Sca-1 antibodies to isolate by cell sorting the ckit+Sca-1+ Lineage negative population (LKS). Staining with relevant isotype controls was included in each sample to determine the right ckit+Sca-1+ population.</p

Differences in LKS cell gene expression.

<p>Fold changes of lineage-specific genes differentially regulated in Lin-ckit+Sca-1+ cells derived from Static and bioreactor culture conditions (Synthecon and Spinner flask) relative to LKS cells purified from BM and FL. Expression levels (n = 3) were compared for genes encoding cell surface markers of hematopoiesis (<b>A, B</b>), and blood cell markers (erythropoiesis) (<b>C,D</b>), the HSC “fingerprint” genes of Chambers et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051944#pone.0051944-Chambers1" target="_blank">[18]</a> (<b>E,F</b>) and critical hematopoietic transcription factors (<b>G,H</b>). The expression levels of hematopoietic markers (<b>A,B</b>) and blood cell markers (erythropoiesis) (<b>C,D</b>) were primarily down-regulated under ESC conditions as compared to BM and FL. When compared to BM and FL, the majority of the HSC fingerprint (<b>E,F</b>) and HSC transcription factor gene expression (<b>G,H</b>) was also down-regulated. (*) represent gene- expression values of statistical significance (p<0.05).</p

Unique Differentiation Profile of Mouse Embryonic Stem Cells in Rotary and Stirred Tank Bioreactors

Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (Synthecon™) type bioreactors. In general, high percentages of hematopoietic stem and progenitor cells were generated in both bioreactors, especially at high cell densities. In addition, Synthecon bioreactors produced more sca-1+ progenitors and spinner flasks generated more c-Kit+ progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that on day 7 of differentiation, embryoid bodies from both bioreactors consisted of all three germ layers of embryonic development. However, unique gene expression profiles were observed in the two bioreactors; for example, expression of specific hematopoietic genes were significantly more upregulated in the Synthecon cultures than in spinner flasks. We conclude that bioreactor type and culture parameters can be used to control ES cell differentiation, enhance unique progenitor cell populations, and provide means for large-scale production of transplantable therapeutic cells

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

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.This work was partially supported through National Institutes of Health grant number EB005026 to KR and PWT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.Biomedical Engineerin