Isolation and Characterization of a Human Fetal Mesenchymal Stem Cell Population: Exploring the Potential for Cell Banking in Wound Healing Therapies

Abstract

Various cell-based therapies are in development to address chronic and acute skin wound healing, for example for burns and trauma patients. An off-the-shelf source of allogeneic dermal cells could be beneficial for innovative therapies accelerating the healing in extensive wounds where the availability of a patient's own cells is limited. Human fetal-derived dermal fibroblasts (hFDFs) show high in vitro division rates, exhibit low immunological rejection properties, and present scarless wound healing in the fetus, and previous studies on human fetal tissue-derived cell therapies have shown promising results on tissue repair. However, little is known about cell lineage stability and cell differentiation during the cell expansion process, required for any potential therapeutic use. We describe an isolation method, characterize a population, and investigate its potential for cell banking and thus suitability as a potential product for cell grafting therapies. Our results show hFDFs and a bone marrow-derived mesenchymal stem cell (BM-MSC) line shared identification markers and in vitro multilineage differentiation potential into osteogenic, chondrogenic, and adipogenic lineages. The hFDF population exhibited similar cell characteristics as BM-MSCs while producing lower pro-inflammatory cytokine IL-6 levels and higher levels of the wound healing factor hepatocyte growth factor. We demonstrate in vitro differentiation of hFDFs, which may be a problem in maintaining long-term lineage stability, potentially limiting their use for cell banking and therapy development.

Keywords: cell banking; cell therapy; fetal-derived MSC; mesenchymal stem cells; wound healing.

Fig. 1.

Human fetal dermal-derived fibroblasts (hFDFs) off-the-shelf concept. Fetal dermal-derived fibroblasts are selected by mechanical disruption and cultured (A). Fibroblast-like cells are selected from other lineages using a stem cell knife (B). Cells are sorted using flow cytometry, cultured for expansion (C), and frozen (D). Conceptually, the cells will be thawed for clinical use applying a cell spray technique (E).

Fig. 2.

Detection of mesenchymal stem cell (MSC) population in human fetal dermal-derived fibroblasts (hFDFs). (A) Adipogenic, chondrogenic and osteogenic differentiation of hFDF cells compared with bone marrow (BM)-MSCs as a positive control. Adipogenic differentiation was induced by IBMX (3-isobutyl-1-methylxanthine), and staining was done with Oil Red O for fatty acids. Chondrogenic differentiation was induced by TGF-β3, and cells were stained with Alcian blue for proteoglycans. Osteogenic differentiation was induced by B-Glycerophosphate, and cells were stained with Alizarin S red for calcium deposition. (B) Flow cytometry chart showing percentages of mesenchymal stem cell antigen markers of passages #1–3 of cultured 9–11-week-old fetal dermal fibroblasts (n=6). Isotypes (red) and human fetal-derived fibroblast signal (blue). Maximum gate error of 5%. (C) Gene expression analysis for markers CD105, CD90, CD73, CD34, CD45 of different hFDF lineages (n=6), comparing passages 2–3 (n=6) with hFDF passages 4–7 (n=4), normalized to bone marrow mesenchymal stromal cells (n=3). (D) Cell growth rate analysis, comparing the doubling time (k) among hFDF donors (n=20) of passages 1–3 and 4–6, with/without sorting and MSC (n=3). ** (p<0.01).

Fig. 3.

Sorted hFDF populations showing differentiation after 6 in vitro passages. (A) Skin biopsy section of 10 weeks gestational age, showing human fetal dermal fibroblasts before isolation. Other cell lineages are visible such as bone (b), dermis (d), and epidermis (e). (B) outgrowth of hFDF cells after isolation. (C) Differentiated hFDF cells at passage 2 expressing α-SMA. (D) Gene expression analysis of MACS CD105^+/− sorted hFDF cells (n=2) compared with the raw population. (E) Gene expression analysis of passages 1 and 2 of MACS CD34⁻/CD45⁻/CD105⁺ sorted hFDF cells (n=2) compared with the raw population. (F) Immunofluorescence staining of MSC-like population surface antigen markers CD105, CD90, CD73 present in hFDF populations before sorting. (G) Sorted hFDF population in vitro during expansion. (H) Flow Cytometry sorting strategy showing mesenchymal stem cell antigen marker isotypes (red) and human fetal-derived fibroblast signals (blue). Max gate error=5%. (I) hFDF population percentage showing MSC antigen markers CD105⁺/CD90⁺/CD73⁺/CD34⁻/CD45⁻/CD14⁻/CD79α⁻/HLA-DR⁻ after sorting (p0) and after 6 weeks (p6) in culture. (J) Flow cytometry analysis is showing changes in percentages of hFDF cells presenting the individual MSC markers CD105, CD90, CD73, CD34 and, CD45 after sorting compared with 6 weeks in culture. (K) Gene expression analysis of MSC markers of hFDF populations between passage 3 (n=3) and 6 (n=3). (D, E, and J) Gene expression results were normalized to mesenchymal stem cell populations, using beta-actin as a housekeeping gene.

Fig. 4.

Cytokine secretion and contraction assay comparing hFDFs and MSCs. (A) Secretion of interleukin 6 (IL-6) and hepatocyte growth factor (HGF) of fetal dermal-derived fibroblasts at passage 3 compared with mesenchymal stromal cells at passage 2. The HGF and IL-6 concentration were normalized to the cell number in the culture at 48 h (pg/ml per well). (B) 10⁵ cells of passage 2 fetal dermal-derived cells or mesenchymal stem cells were mixed with collagen I matrix. After 48 h in culture, cells mixed with collagen matrix were physically detached from the edge of the well and the discs were analyzed for matrix contraction. Results show the contraction after 20 h. 2,3 butanedione monoxime (BDM) was used to inhibit contraction (control). (C) Contraction is given as area; the same samples, cultured with (BDM) or without (control) inhibitor for contraction. (D) Detail of the cells and collagen matrix disc edge.

Fig. 5.

Gene expression of cultured cells in collagen I matrix. (A) Gene expression analyses of hFDF and MSC donors embedded in collagen I matrix (3D culture) compared with their correspondent 2D cultured homologs (controls). (B) Gene expression analysis of different hFDF cell lineages cultured in 2D compared with MSCs as a control. Data are given as mean from three technical repeats ± standard error. Results are presented as ΔΔCt mean, normalized to beta-actin housekeeping gene expression. ELN: Elastin; Col 1A: Collagen 1A; Col2B: Collagen 2B; HGF: Hepatocyte growth factor; IL-6: Interleukine-6; ki67: proliferation protein.

Fig. 6.

Scratch assay to test wound healing capabilities measuring the cell migration after 24 and 48 h. The open wound area is represented by the percentage of scratched surface without cell coverage.