Regeneration of Articular Cartilage by Human ESC-Derived Mesenchymal Progenitors Treated Sequentially with BMP-2 and Wnt5a

Abstract

The success of cell-based therapies to restore joint cartilage requires an optimal source of reparative progenitor cells and tight control of their differentiation into a permanent cartilage phenotype. Bone morphogenetic protein 2 (BMP-2) has been extensively shown to promote mesenchymal cell differentiation into chondrocytes in vitro and in vivo. Conversely, developmental studies have demonstrated decreased chondrocyte maturation by Wingless-Type MMTV Integration Site Family, Member 5A (Wnt5a). Thus, we hypothesized that treatment of human embryonic stem cell (hESC)-derived chondroprogenitors with BMP-2 followed by Wnt5a may control the maturational progression of these cells into a hyaline-like chondrocyte phenotype. We examined the effects of sustained exposure of hESC-derived mesenchymal-like progenitors to recombinant Wnt5a or BMP-2 in vitro. Our data indicate that BMP-2 promoted a strong chondrogenic response leading to terminal maturation, whereas recombinant Wnt5a induced a mild chondrogenic response without promoting hypertrophy. Moreover, Wnt5a suppressed BMP-2-mediated chondrocyte maturation, preventing the formation of fibrocartilaginous tissue in high-density cultures treated sequentially with BMP-2 and Wnt5a. Implantation of scaffoldless pellets of hESC-derived chondroprogenitors pretreated with BMP-2 followed by Wnt5a into rat chondral defects induced an articular-like phenotype in vivo. Together, the data establish a novel role for Wnt5a in controlling the progression from multipotency into an articular-like cartilage phenotype in vitro and in vivo. Stem Cells Translational Medicine 2017;6:40-50.

Keywords: Articular cartilage; BMP-2; Embryonic stem cells; Mesenchymal stem cells; Wnt5a.

Figure 1

Mesenchymal stem cell‐like progenitors derived from H9 hESCs (H9‐derived MSCs) share common features with bone marrow‐derived MSCs. (A): Expression of cell surface antigens on progenitor cells by flow cytometry analysis. Histograms display percent cells expressing cell‐surface antigens of mesenchymal markers on H9‐derived MSC‐like cells (H9‐MSCs). H9‐MSCs express markers associated with the mesenchymal phenotype (CD44, CD73, CD29, CD166, CD90, CD105, and HLA‐ABC) and lack expression of endothelial (CD31) and hematopoietic markers (CD45 and HLA‐DR). Negative isotype controls are shown with percent fluorescence indicated for positive signal over isotype controls. (B): Comparative flow cytometry analyses of H9‐derived MSCs and the adult human bone marrow‐derived MSCs (Lonza) showed similar cell surface expression profiles. (C): Quantitative reverse‐transcriptase polymerase chain reaction analyses revealed downregulation of canonical pluripotency genes in H9‐derived MSC progenitors compared with undifferentiated H9 ESCs. (D): Alcian Blue staining of H9‐derived MSC high‐density pellets cultured in serum‐free chondrogenic medium for 21 days (magnification, ×4); Oil Red O staining of H9‐derived MSC progenitor monolayer cells cultured in adipogenic medium for 21 days (magnification, ×20); and a 10‐cm tissue culture dish (magnification, ×1) with alkaline phosphatase staining of H9‐derived MSC monolayer cells cultured in osteogenic medium for 21 days. Abbreviations: ALP, alkaline phosphatase; BM‐MSC, bone marrow‐derived mesenchymal stem cell; ESC, embryonic stem cell; FACS, fluorescence‐activated cell sorting; FITC‐A, fluorescein isothiocyanate A; hESC, human embryonic stem cell; HLA‐ABC, human leukocyte antigen ABC; Max, maximum; MSC, mesenchymal stem cell; PE‐A, phycoerythrin A.

Figure 2

Wnt5a promoted chondrogenesis and limited hypertrophy in H9‐derived mesenchymal stem cell (MSC) pellets. (A): Analysis of H9‐MSC progenitors cultured as high‐density pellets for up to 14 days in serum‐free chondrogenic medium with or without BMP‐2 (100 ng/mL) or Wnt5a (50 ng/mL). Safranin O staining of 5‐μm histological sections of growth factor‐treated pellets at days 5, 7, 10, and 14 showed proteoglycan deposition in both the BMP‐2‐ and the Wnt5a‐treated samples. Quantification of percent area stained with Safranin O (n = 6) is shown for each time point recorded. Scale bars = 500 μm. (B): Alcian Blue staining with nuclear fast red counterstain showed presence of hypertrophic chondrocytes in BMP‐2‐treated pellets, which were not detected in the Wnt5a‐treated pellets.

Figure 3

Wnt5a induced chondrogenic gene expression and limited expression of the hypertrophic chondrocyte markers in H9‐derived MSC pellets. Comparative expression of early chondro‐progenitor genes SOX9 (A) and COL2A1 (B) and the expression of mature chondrocyte markers of hypertrophy COL10A1 (C) and ALP (D) in H9‐derived MSC pellets cultured for 14 days with or without 100 ng/ml BMP‐2 or 50 ng/ml Wnt5a. Values greater than 1.0 represent a fold‐change increase in gene expression and less than a 1.0 relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p < .05 (treatment compared with untreated controls); #, p < .05 (statistical significance between treatment groups). Abbreviations: ALP, alkaline phosphatase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Figure 4

Sequential treatment with BMP‐2 followed by Wnt5a induces sustained expression of chondrocyte matrix markers under chondrogenic conditions. (A): Schematic shows treatment conditions for in vitro chondrogenic differentiation of H9‐MSCs. (B–E): Expression data based on quantitative polymerase chain reaction analyses of cartilage matrix genes in treatment groups from differentiating H9‐derived mesenchymal stem cell (MSC) pellets cultured for 5 days in 100 ng/ml BMP‐2 followed by up to 9 days of no growth factor treatment (5D BMP2‐Alone) or sequential treatment with 50 ng/ml Wnt5a (5D BMP2‐Wnt5a): ratio of COL2A1:COL1A1 (B), ACAN (C), COL11A1 (D), and expression of COL9A1 (E). Values greater than 1.0 represent a fold‐change increase in gene expression, and those less than 1.0 indicate a relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p < .05 (treatment compared with untreated controls); #, p < .05 (statistical significance between treatment groups). Abbreviation: D, day.

Figure 5

Wnt5a suppressed BMP‐2‐induced markers of chondrocyte hypertrophy. Comparative expression of mature chondrocyte markers of hypertrophy COL10A1 (A) and ALP (B) in H9‐derived mesenchymal stem cells (MSCs) cultured for 5 days in 100 ng/ml BMP‐2, followed by up to 9 days of no growth factor treatment (5D BMP2‐Alone) or sequential treatment with 50 ng/ml Wnt5a (5D BMP2‐Wnt5a). Values greater than 1.0 represent a fold‐change increase in gene expression and less than 1.0 indicate a relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p < .05 (treatment compared with untreated controls); #, p < .05 (statistical significance between treatment groups). Abbreviations: ALP, alkaline phosphatase; 5D, 5 days.

Figure 6

Representative gross morphometric images (magnification, ×10) and collagen type II immunostaining demonstrate enhanced cartilage repair mediated by H9‐derived mesenchymal stem cell (MSC) pellets pretreated with sequential BMP‐2 and Wnt5a in the rat chondral defect model at 8 weeks. Scale bars = 500 μm. (A, B): Toluidine blue and Fast Green staining of untreated (A) and pretreated (2 days of BMP‐2 followed by 12 days of Wnt5a) implanted H9‐derived MSC pellets (B) at the defect site showed enhanced staining for the treatment group. (C, D): Collagen type II immunohistochemistry of the untreated (C) and sequentially treated pellets (D) demonstrates significant staining for the treatment group and the absence of staining for the untreated control. (E, F): Mean histological scores obtained using a published grading scale for cartilage repair at 4 week (E) and 8 weeks (F) 46. ∗, Total histological scores of chondral defect repair exhibited significance of chondral repair in the pretreatment group over the untreated group at 8 weeks.

Figure 7

Regeneration of permanent cartilage‐like tissue by implanted H9‐derived mesenchymal stem cells (MSCs) pretreated with sequential BMP‐2 and Wnt5a. Quantitative reverse‐transcriptase polymerase chain reaction of preamplified tissue scraped from the defected regions on prepared slides of paraffin‐embedded rat knees 4 and 8 weeks after surgery is shown. Human‐specific expressions of the early chondrogenic gene marker COL2A1 and articular chondrocyte matrix markers COL11A1, ACAN, and PRG4 were detected in only in tissue scrapings from animals receiving the untreated human H9‐derived MSCs, and the human H9‐derived MSCs pretreated for 14 days (2 days, BMP‐2, 12 days, Wnt5a). Values greater than 1.0 represent a fold‐change increase in gene expression and less than 1.0 indicate a relative decrease in expression in comparison with the undifferentiated H9‐derived MSCs (day 0). ∗, p < .05. Abbreviation: ND, no detection of genes in host rat cartilage tissue using human‐specific gene primers.