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The ability of human stem cells to generate somatic cell lineages makes them ideal candidates for use in toxicological testing and eventually, preclinical drug development. Such resources would support an evolution away from human primary cells or research animal models, which suffer from variability and poor predictability, toward off-the-shelf assays of chemical toxicity and drug efficacy using human cells and tissues. To this end, we generated vascular cell populations (smooth muscle cells and endothelial cells) from human pluripotent stem cells (hPSCs), arranged them into 3D co-cultures within supportive gel matrices, and directed their propensity for self-organization resembling microvasculature. The resulting vascular cell populations and co-cultured constructs were then arrayed in high throughput and used for screening a library of environmental and clinical chemical agents for immunological and toxicological responses. The screen effectively stratified the chemicals into various levels of toxicity, with both cell type-specific and co-culture-dependent responses observed. Thus, hPSC-derived vascular cells and constructs could be progressed further toward use in toxicant and drug screening.

Differentiation of mesenchymal stromal/stem cells (MSCs) involves a series of molecular signals and gene transcription events required for attaining cell lineage commitment. Modulation of the actin cytoskeleton using cytochalasin D (CytoD) drives osteogenesis at early timepoints in bone marrow-derived MSCs and also initiates a robust osteogenic differentiation program in adipose tissue-derived MSCs. To understand the molecular basis for these pronounced effects on osteogenic differentiation, we investigated global changes in gene expression in CytoD-treated murine and human MSCs by high-resolution RNA-sequencing (RNA-seq) analysis. A three-way bioinformatic comparison between human adipose tissue-derived MSCs (hAMSCs), human bone marrow-derived MSCs (hBMSCs), and mouse bone marrow-derived MSCs (mBMSCs) revealed significant upregulation of genes linked to extracellular matrix organization, cell adhesion and bone metabolism. As anticipated, the activation of these differentiation-related genes is accompanied by a downregulation of nuclear and cell cycle-related genes presumably reflecting cytostatic effects of CytoD. We also identified eight novel CytoD activated genes-VGLL4, ARHGAP24, KLHL24, RCBTB2, BDH2, SCARF2, ACAD10, HEPH-which are commonly upregulated across the two species and tissue sources of our MSC samples. We selected the Hippo pathway-related VGLL4 gene, which encodes the transcriptional co-factor Vestigial-like 4, for further study because this pathway is linked to osteogenesis. VGLL4 small interfering RNA depletion reduces mineralization of hAMSCs during CytoD-induced osteogenic differentiation. Together, our RNA-seq analyses suggest that while the stimulatory effects of CytoD on osteogenesis are pleiotropic and depend on the biological state of the cell type, a small group of genes including VGLL4 may contribute to MSC commitment toward the bone lineage.

Insufficient cell number hampers therapies utilizing adult human mesenchymal stem cells (hMSCs) and current ex vivo expansion strategies lead to a loss of multipotentiality. Here we show that supplementation with an embryonic form of heparan sulfate (HS-2) can both increase the initial recovery of hMSCs from bone marrow aspirates and increase their ex vivo expansion by up to 13-fold. HS-2 acts to amplify a subpopulation of hMSCs harboring longer telomeres and increased expression of the MSC surface marker stromal precursor antigen-1. Gene expression profiling revealed that hMSCs cultured in HS-2 possess a distinct signature that reflects their enhanced multipotentiality and improved bone-forming ability when transplanted into critical-sized bone defects. Thus, HS-2 offers a novel means for decreasing the expansion time necessary for obtaining therapeutic numbers of multipotent hMSCs without the addition of exogenous growth factors that compromise stem cell fate.

While defining the environment for human embryonic stem cell (hESC) culture on 2-dimensional (2D) surfaces has made rapid progress, the industrial-scale implementation of this technology will benefit from translating this knowledge into a 3-dimensional (3D) system, thus enabling better control, automation, and volumetric scale-up in bioreactors. The current study describes a system with defined conditions that are capable of supporting the long-term 2D culture of hESCs and the transposing of these conditions to 3D microcarrier (MC) cultures. Vitronectin (VN) and laminin (LN) were chosen as matrices for the long-term propagation of hESCs in a defined culture medium (STEMPRO(®)) for conventional 2D culture. Adsorption of these proteins onto 2D tissue culture polystyrene (TCPS) indicated that surface density saturation of 510 and 850 ng/cm(2) for VN and LN, respectively, was attained above 20 μg/mL deposition solution concentration. Adsorption of these proteins onto spherical (97±10 μm), polystyrene MC followed a similar trend and coating surface densities of 450 and 650 ng/cm(2) for VN and LN, respectively, were used to support hESC propagation. The long-term expansion of hESCs was equally successful on TCPS and MC, with consistently high expression (>90%) of pluripotent markers (OCT-4, MAB-84, and TRA-1-60) over 20 passages and maintenance of karyotypic normality. The average fold increase in cell numbers on VN-coated MC per serial passage was 8.5±1.0, which was similar to LN-coated MC (8.5±0.9). Embryoid body differentiation assays and teratoma formation confirmed that hESCs retained the ability to differentiate into lineages of all 3 germ layers, thus demonstrating the first translation to a fully defined MC-based environment for the expansion of hESCs.

The growth and differentiation of mesenchymal stem cells (MSCs) is controlled by various growth factors, the activities of which can be modulated by heparan sulfates (HSs). We have previously noted the necessity of sulfated glycosaminoglycans for the fibroblast growth factor type 2 (FGF-2)-stimulated differentiation of osteoprogenitor cells. Here we show that exogenous application of HS to cultures of primary rat MSCs stimulates their proliferation, leading to increased expression of osteogenic markers and enhanced bone nodule formation. FGF-2 can also increase the proliferation, and osteogenic differentiation of rat bone marrow stem cells (rMSCs) when applied exogenously during their linear growth. However, as opposed to exogenous HS, the continuous use of FGF-2 during in vitro differentiation completely blocked rMSC mineralization. We show that the effects of both FGF-2 and HS are mediated through FGF receptor 1 (FGFR1) and that inhibition of signaling through this receptor arrests cell growth, resulting in the cells being unable to reach the critical density necessary to induce differentiation. Blocking FGFR1 signaling in postconfluent osteogenic cultures significantly increased calcium deposition. Taken together our data suggest that FGFR1 signaling plays an important role during osteogenic differentiation, first by stimulating cell growth that is closely followed by an inhibitory effect once the cells have reached confluence. It also confirms the importance of HS as a coreceptor for the signaling of endogenous FGF-2 and suggests that purified glycosaminoglycans may be attractive alternatives to growth factors for improved ex vivo growth and differentiation of MSCs.

Adult human mesenchymal stem cells (hMSCs) are able to differentiate into a range of specific cell types in vitro and in vivo, and thus hold tremendous potential for use in regenerative medicine. Despite this promise, deficient understanding of the mechanisms that regulate their differentiation has precluded their widespread use. Genetic manipulation of hMSCs by introduction of transgenes is an indispensable tool for gaining insight into these mechanisms. Like most primary cultures, hMSCs are difficult to transfect with conventional techniques, and although some viral transduction techniques are highly efficient, the protocols require extensive optimization and contain significant health risks. We were generally unable to achieve high transfection efficiencies with lipofection-based reagents that we found, in contrast to electroporation, adversely affected hMSC proliferation and differentiation. Here we report a simple and reliable electroporation protocol that results in transfection efficiencies up to 90% that are comparable to most viral methods while maintaining hMSC stemness. Most importantly, our protocol does not rely on a specific electroporator with preset programs and unique buffers, and is thus much simpler, cheaper, and easier to optimize. Furthermore, we show sustained transgene expression lasting several weeks that was useful for assessing the effects on hMSC function and in transient expression gene therapy.

The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.

Therapeutic modalities aimed at bone regeneration are increasingly employing extracellular matrix (ECM) constituents to control bone marrow progenitor cell (BMPC) commitment, growth, and differentiation. However, the precise role these ECM elements play during stem cell differentiation remains unclear. (See also Salaszynk et al., Stem Cells Dev 14(6):608-620, 2005; and Schwartz et al., Stem Cells Dev. 14(6), 643-655, 2005, both in this issue.) Because bone formation ultimately begins with the recruitment and commitment of BMPCs into the osteogenic lineage, factors that enhance this process are clearly therapeutic targets. We hypothesized that BMPC attachment, proliferation, and osteogenic differentiation would be potentiated when cultured on ECM proteins normally found in the bone niche. To examine this, we cultured murine BMPCs on laminin-1, fibronectin, and collagen type-1 substrates for up to 14 days and assessed their homogeneity, attachment, proliferation, and expression of the specific bone lineage markers RUNX2, collagen-1, alkaline phosphatase, and osteocalcin. We found that freshly harvested mBMPCs contain a mixed population of progenitor cells and that the mesenchymal pool can be enriched by adherent culture in the presence of leucine methyl ester. Furthermore, mBMPCs attached to laminin, fibronectin, and collagen-1 with varying affinity up to 3 h (fibronectin>or=collagen>laminin), after which time no difference could be detected. Despite this, growth was unaffected; cells thereafter proliferated equally well on all substrates up to confluence (7 days). Notably, commitment to the osteoblast lineage (RUNX2) increased up to 14 days for cells cultured on the various substrates, yet no difference was observed at day 14 in the expression of collagen-1, alkaline phosphatase, or osteocalcin. We conclude that mBMPC differentiation down the osteoblastic lineage is time-dependent in osteogenic culture and that attachment to ECM matrices potentiates lineage commitment rather than growth.