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. 2011 Feb 21;2(1):10.
doi: 10.1186/scrt51.

Fibroblasts derived from human embryonic stem cells direct development and repair of 3D human skin equivalents

Yulia Shamis  1 , Kyle J Hewitt, Mark W Carlson, Mariam Margvelashvilli, Shumin Dong, Catherine K Kuo, Laurence Daheron, Christophe Egles, Jonathan A Garlick
Affiliations

Fibroblasts derived from human embryonic stem cells direct development and repair of 3D human skin equivalents

Yulia Shamis et al. Stem Cell Res Ther. .

Abstract

Introduction: Pluripotent, human stem cells hold tremendous promise as a source of progenitor and terminally differentiated cells for application in future regenerative therapies. However, such therapies will be dependent upon the development of novel approaches that can best assess tissue outcomes of pluripotent stem cell-derived cells and will be essential to better predict their safety and stability following in vivo transplantation.

Methods: In this study we used engineered, human skin equivalents (HSEs) as a platform to characterize fibroblasts that have been derived from human embryonic stem (hES) cell. We characterized the phenotype and the secretion profile of two distinct hES-derived cell lines with properties of mesenchymal cells (EDK and H9-MSC) and compared their biological potential upon induction of differentiation to bone and fat and following their incorporation into the stromal compartment of engineered, HSEs.

Results: While both EDK and H9-MSC cell lines exhibited similar morphology and mesenchymal cell marker expression, they demonstrated distinct functional properties when incorporated into the stromal compartment of HSEs. EDK cells displayed characteristics of dermal fibroblasts that could support epithelial tissue development and enable re-epithelialization of wounds generated using a 3D tissue model of cutaneous wound healing, which was linked to elevated production of hepatocyte growth factor (HGF). Lentiviral shRNA-mediated knockdown of HGF resulted in a dramatic decrease of HGF secretion from EDK cells that led to a marked reduction in their ability to promote keratinocyte proliferation and re-epithelialization of cutaneous wounds. In contrast, H9-MSCs demonstrated features of mesenchymal stem cells (MSC) but not those of dermal fibroblasts, as they underwent multilineage differentiation in monolayer culture, but were unable to support epithelial tissue development and repair and produced significantly lower levels of HGF.

Conclusions: Our findings demonstrate that hES-derived cells could be directed to specified and alternative mesenchymal cell fates whose function could be distinguished in engineered HSEs. Characterization of hES-derived mesenchymal cells in 3D, engineered HSEs demonstrates the utility of this tissue platform to predict the functional properties of hES-derived fibroblasts before their therapeutic transplantation.

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Figures

Figure 1
Figure 1
Characterization of hES-derived mesenchymal cell lines. (a) Schematic summarizing the two protocols used for generating EDK and H9-MSC from H9 human embryonic stem (hES) cells. (b) Cellular morphology of human foreskin fibroblasts (HFF), EDK, and H9-MSC using phase contrast. Bars, 50 μm. (c) Expression of mesenchymal markers vimentin, Thy1, and α-SMA following immunofluorescence staining. Bars, 50 μm. (d) Comparison of surface antigen expression on HFF, EDK, and H9-MSC by flow cytometric analysis. EDK and H9-MSCs express markers associated with mesenchymal phenotype (positive for expression CD73, CD105, CD44, CD13, CD106, and CD166; negative for expression of CD45, CD34, and CD31). EDK and H9-MSCs show significant differences in expression of CD146 and CD10 antigens. (HFF, filled grey profile; EDK, red profile; H9-MSC, black profile).
Figure 2
Figure 2
Adipogenic and osteogenic differentiation of hES-derived mesenchymal cells. (a) Analysis of osteogenic activity of EDK, H9-MSC, and human foreskin fibroblasts (HFF). Osteogenic differentiation was induced using the β-glycerophosphate method, after four weeks of differentiation, calcium deposition was stained with alzarin red. Bars, 50 μm. (b) Quantification of alizarin red staining (t-test: **P < 0.01). Results are expressed as the mean +/- standard deviation (SD) of three independent experiments and three technical replicates per experiment. (c) Analysis of adipogenic activity of EDK, H9-MSC, and HFF. Adipogenic differentiation was induced using the 3-isobutyl-1-methylxanthine (IBMX) method, and after four weeks of differentiation the lipid vesicles were stained with oil red O. Bars, 50 μm. (d) Quantification of oil red O staining (t-test: **P < 0.01). Results are expressed as the mean +/- SD of three experiments and three technical replicates per experiment.
Figure 3
Figure 3
EDK cells but not H9-MSC promote epidermal morphogenesis in HSE tissues. (a): Tissue morphology of human skin-equivalent (HSE) tissues constructed using human foreskin fibroblasts (HFF), EDK, H9-MSC, or without cells as analyzed by H&E staining. (b) The expression pattern of the epidermal differentiation marker keratin 1/10 was analyzed by immunofluorescence (red). Bars, 50 μm. (c) Comparison of the secretory profile of EDK, H9-MSC, and HFF cells by antibody-based cytokine array (d) Histogram profiles generated by quantifying the mean spot pixel densities by ImageJ from the array membrane shown above. The data are presented as percentages of the respective positive controls. (e) Hepatocyte growth factor (HGF) mRNA expression in HFF, EDK, and H9-MSC by real-time RT-PCR. Data are normalized to GAPDH, and all results are expressed as the mean +/- standard deviation (SD) of three experiments and three technical replicates per experiment. (f) Levels of HGF in supernatants from HFF, EDK, and H9-MSC monolayer cultures by ELISA. Data are normalized per 104 cells and all results are expressed as the mean +/- SD of three experiments and three technical replicates per experiment.
Figure 4
Figure 4
EDK cells accelerate re-epithelialization of wounded HSEs. (a) Schematic of 3D tissue model of wound re-epithelialization. In step 1, a full-thickness wound is generated by excising a human skin-equivalent (HSE). In step 2, the wounded HSE is placed on a second, contracted collagen gel populated with EDK, H9-MSC, human foreskin fibroblasts (HFF), or constructed without cells. In step 3, keratinocytes (NHK) undergo migration to close the wound gap and restored epithelial integrity. The far right panel is an image of six-well insert containing HSE 96 hours after wounding. (b) Representative morphology of wounded tissues constructed with HFF, EDK, H9-MSC, or without cells 96 hours after wounding (black arrows demarcate the initial wound edges). Bars, 200 μm. (c) EDK showed a rate of wound closure similar to tissues in which HFF were incorporated. The degree of re-epithelialization was significantly lower in H9-MSC and no cells-containing tissues as compared with HFF (t-test: **P < 0.01). (d) Levels of hepatocyte growth factor (HGF) in supernatants of wounded tissues 96 hours after wounding as measured by ELISA. HFF- and EDK-containing tissues produced higher levels of HGF as compared with H9-MSC-containing tissues or tissues constructed without cells. All results are presented as the mean +/- standard deviation of three independent experiments and three technical replicates.
Figure 5
Figure 5
Suppression of HGF production impairs EDK-mediated re-epithelialization of wounded HSEs. (a) Efficiency of shRNA knockdown of hepatocyte growth factor (shHGF) in EDK cells relative to a scrambled, shRNA control (shScram) as measured by ELISA. Secretion of hepatocyte growth factor (HGF; top panel) was reduced 95% relative to shScram (t-test: **P < 0.01), secretion of KGF (lower panel) was not affected by shHGF. Data are normalized to shScram and all results are expressed as the mean +/- standard deviation (SD) of three experiments and three technical replicates per experiment. (b) Representative morphology of wounded tissues constructed with EDK-shHGF and EDK-shScram cells 72 hours after wounding (black arrows demarcate the initial wound edges; white arrows demarcate the tip of epithelial tongues). Bars, 200 μm. (c) The degree of re-epithelialization was significantly lower in tissues containing EDK-shHGF cells as compared with EDK-shScram (t-test: **P < 0.01). (d) Relative levels of HGF in supernatants of wounded tissues 72 hours after wounding as measured by ELISA. Tissues containing EDK-shHGF cells produced significantly lower levels of HGF as compared with EDK-shScram (t-test: **P < 0.01). (e) Proliferation of basal keratinocytes in tissues containing EDK-shHGF and EDK-shScram cells was analyzed using immunoperoxidase staining with anti-BrdU antibody 72 hours after wounding (black arrows demarcate the initial wound edges; white arrows demarcate the BrdU-positive cells). (f) Quantification of BrdU-positive basal keratinocytes. Tissues containing EDK-shHGF demonstrated significantly lower percentage of proliferating basal keratinocytes compared with EDK-shScram (t-test: **P < 0.01). Bars, 100 μm. All results are presented as the mean +/- SD of three independent experiments and three technical replicates.
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