Roberto Narcisi

Long-Term Expansion, Enhanced Chondrogenic Potential, and Suppression of Endochondral Ossification of Adult Human MSCs via WNT Signaling Modulation

Summary Mesenchymal stem cells (MSCs) are a potential source of chondrogenic cells for the treatment of cartilage disorders, but loss of chondrogenic potential during in vitro expansion and the propensity of cartilage to undergo hypertrophic maturation impede their therapeutic application. Here we report that the signaling protein WNT3A, in combination with FGF2, supports long-term expansion of human bone marrow-derived MSCs. The cells retained their chondrogenic potential and other phenotypic and functional properties of multipotent MSCs, which were gradually lost in the absence of WNT3A. Moreover, we discovered that endogenous WNT signals are the main drivers of the hypertrophic maturation that follows chondrogenic differentiation. Inhibition of WNT signals during differentiation prevented calcification and maintained cartilage properties following implantation in a mouse model. By maintaining potency during expansion and preventing hypertrophic maturation following differentiation, the modulation of WNT signaling removes two major obstacles that impede the clinical application of MSCs in cartilage repair.

Silencing of Antichondrogenic MicroRNA-221 in Human Mesenchymal Stem Cells Promotes Cartilage Repair In Vivo

There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering‐based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation toward the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the antichondrogenic regulator microRNA‐221 (miR‐221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF‐β. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR‐221 silencing was sufficient to drive hMSCs toward chondrogenic differentiation in the absence of TGF‐β. In vivo, the potential of miR‐221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR‐221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate‐only). Notably, miR‐221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR‐221 has a prochondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. Stem Cells 2016;34:1801–1811

TGFβ inhibition during expansion phase increases the chondrogenic re-differentiation capacity of human articular chondrocytes

OBJECTIVE Autologous chondrocyte implantation is a cell-based treatment to repair articular cartilage defects, relying on the availability of expanded (de-differentiated) chondrocytes. Unfortunately, the expansion process causes several phenotypical changes, requiring re-establishment of the native chondrogenic phenotype to sustain proper repair. Among other proteins, transforming growth factor-β (TGFβ) is known to influence the chondrogenic re-differentiation of human articular chondrocytes (HACs) and their matrix deposition. Thus we investigated the effects of TGFβ-depletion during the expansion phase. DESIGN HACs were isolated from articular cartilage and expanded in the canonical serum-supplemented medium [fetal calf serum (FCS)] or in a chemically-defined (CD) medium, with or without anti-TGFβ antibody administration. The re-differentiation potential of the cells was assessed by pellet cultures, gene expression analysis and histology. RESULTS Cell proliferation proceeded more rapidly in CD-medium than in FCS-medium; it was not affected by the use of anti-TGFβ antibody but was further increased by addition of exogenous TGFβ1, via increased p-Smad1/5/8. Conversely, in FCS-medium, addition of anti-TGFβ antibody decreased both proliferation and p-Smad1/5/8 level. Challenging either FCS- or CD-medium with anti-TGFβ antibody during expansion enhanced chondrogenesis in the subsequent pellet cultures. Moreover, TGFβ-depletion during expansion in CD-medium inhibited mRNA expression of hypertrophic markers, collagen type-X (COL10) and matrix metalloproteinase-13 (MMP-13). Interestingly, the TGFβ1 level detected by enzyme-linked immunosorbent sandwich assay (ELISA) during cell expansion was correlated with COL10 mRNA expression after re-differentiation. CONCLUSION TGFβ-depletion during expansion improves the re-differentiation capacity of chondrocytes and inhibits hypertrophy. These results indicate the importance of the expansion medium composition to improve chondrogenic re-differentiation and to inhibit hypertrophy.

FGF, TGFβ and Wnt crosstalk: embryonic to in vitro cartilage development from mesenchymal stem cells

Articular cartilage is easily damaged, yet difficult to repair. Cartilage tissue engineering seems a promising therapeutic solution to restore articular cartilage structure and function, with mesenchymal stem cells (MSCs) receiving increasing attention for their promise to promote cartilage repair. It is known from embryology that members of the fibroblast growth factor (FGF), transforming growth factor‐β (TGFβ) and wingless‐type (Wnt) protein families are involved in controlling different differentiation stages during chondrogenesis. Individually, these pathways have been extensively studied but so far attempts to recapitulate embryonic development in in vitro MSC chondrogenesis have failed to produce stable and functioning articular cartilage; instead, transient hypertrophic cartilage is obtained. We believe a better understanding of the simultaneous integration of these factors will improve how we relate embryonic chondrogenesis to in vitro MSC chondrogenesis. This narrative review attempts to define current knowledge on the crosstalk between the FGF, TGFβ and Wnt signalling pathways during different stages of mesenchymal chondrogenesis. Connecting embryogenesis and in vitro differentiation of human MSCs might provide insights into how to improve and progress cartilage tissue engineering for the future. Copyright

Differential gene expression of the intermediate and outer interzone layers of developing articular cartilage in murine embryos.

Nascent embryonic joints, interzones, contain a distinct cohort of progenitor cells responsible for the formation of the majority of articular tissues. However, to date the interzone has largely been studied using in situ analysis for candidate genes in the context of the embryo rather than using an unbiased genome-wide expression analysis on isolated interzone cells, leaving significant controversy regarding the exact role of the intermediate and outer interzone layers in joint formation. Therefore, in this study, using laser capture microdissection (three biological replicates), we selectively harvested the intermediate and outer interzones of mouse embryos at gestational age 15.5 days, just prior to cavitation, when the differences between the layers should be most profound. Microarray analysis (Agilent Whole Mouse Genome Oligo Microarrays) was performed and the differential gene expression between the intermediate interzone cells and outer interzone cells was examined by performing a two-sided paired Students t-test and pathway analysis. One hundred ninety-seven genes were differentially expressed (≥ 2-fold) between the intermediate interzone and the outer interzone with a P-value ≤ 0.01. Of these, 91 genes showed higher expression levels in the intermediate interzone and 106 were expressed higher in the outer interzone. Pathway analysis of differentially expressed genes suggests an important role for inflammatory processes in the interzone layers, especially in the intermediate interzone, and hence in joint and articular cartilage development. The high representation of genes relevant to chondrocyte hypertrophy and endochondral ossification in the outer interzone suggests that it undergoes endochondral ossification.

Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.

Introduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. Materials & Methods ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. Results ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis. Conclusion ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by mesenchymal stem cells will depend on activation of both receptors.

SMAD3 and SMAD4 have a more dominant role than SMAD2 in TGFβ-induced chondrogenic differentiation of bone marrow-derived mesenchymal stem cells

To improve cartilage formation by bone marrow-derived mesenchymal stem cells (BMSCs), the signaling mechanism governing chondrogenic differentiation requires better understanding. We previously showed that the transforming growth factor-β (TGFβ) receptor ALK5 is crucial for chondrogenesis induced by TGFβ. ALK5 phosphorylates SMAD2 and SMAD3 proteins, which then form complexes with SMAD4 to regulate gene transcription. By modulating the expression of SMAD2, SMAD3 and SMAD4 in human BMSCs, we investigated their role in TGFβ-induced chondrogenesis. Activation of TGFβ signaling, represented by SMAD2 phosphorylation, was decreased by SMAD2 knockdown and highly increased by SMAD2 overexpression. Moreover, TGFβ signaling via the alternative SMAD1/5/9 pathway was strongly decreased by SMAD4 knockdown. TGFβ-induced chondrogenesis of human BMSCs was strongly inhibited by SMAD4 knockdown and only mildly inhibited by SMAD2 knockdown. Remarkably, both knockdown and overexpression of SMAD3 blocked chondrogenic differentiation. Chondrogenesis appears to rely on a delicate balance in the amount of SMAD3 and SMAD4 as it was not enhanced by SMAD4 overexpression and was inhibited by SMAD3 overexpression. Furthermore, this study reveals that TGFβ-activated phosphorylation of SMAD2 and SMAD1/5/9 depends on the abundance of SMAD4. Overall, our findings suggest a more dominant role for SMAD3 and SMAD4 than SMAD2 in TGFβ-induced chondrogenesis of human BMSCs.

Expression of CD105 on expanded mesenchymal stem cells does not predict their chondrogenic potential

OBJECTIVE Total bone marrow-derived mesenchymal stem cell (BMSC) populations differ in their potential to undergo chondrogenesis, with individual BMSCs differing in their chondrogenic capacity. The aim of this study was to explore the use of CD105 as a marker to isolate a chondrogenic subpopulation of BMSCs from the total, heterogeneous population. DESIGN BMSCs were isolated from patients undergoing total hip replacement and following expansion (Passage 1-Passage 5), CD105 expression was investigated by FACS analysis. FACS was also used to sort BMSCs based on the presence of CD105 (CD105(+)/CD105(-)) or their amount of CD105 expression (CD105(Bright)/CD105(Dim)). After 3 or 5 weeks of differentiation, chondrogenic potential was determined by thionine staining for glycosaminoglycan (GAG) content and by detection of collagen type II using immunohistochemistry. RESULTS Expanded total BMSC populations were composed almost exclusively of CD105(+) cells, the percentage of which did not correlate to subsequent chondrogenic potential; chondrogenic potential was observed to diminish with culture although CD105 expression remained stable. Similarly, differences in chondrogenic potential were observed between donors despite similar levels of CD105(+) BMSCs. Comparison of CD105(Bright) and CD105(Dim) BMSCs did not reveal a subpopulation with superior chondrogenic potential. CONCLUSIONS Chondrogenic potential of BMSCs is often linked to CD105 expression. This study demonstrates that CD105 expression on culture expanded BMSC populations does not associate with a chondroprogenitor phenotype and CD105 should not be pursued as a marker to obtain a chondroprogenitor population from BMSCs.

Recombinant human type II collagen hydrogel provides a xeno-free 3D micro-environment for chondrogenesis of human bone marrow-derived mesenchymal stromal cells

Recombinant human type II collagen (rhCII) hydrogel was tested as a xeno‐free micro‐environment for the chondrogenesis of human bone marrow‐derived mesenchymal stromal cells (BM‐MSCs). The rhCII hydrogels were seeded with BM‐MSCs and cultured in a xeno‐free chondro‐inductive medium for 14, 28 and 84 days. High‐density pellet cultures served as controls. The samples were subjected to biochemical, histological and gene expression analyses. Although the cells deposited glycosaminoglycans into the extracellular space significantly more slowly in the rhCII hydrogels compared to the high‐density pellets, a similar potential of matrix deposition was reached by the end of the 84‐day culture. At day 28 of culture, the gene expression level for cartilage marker genes (i.e. genes encoding for Sox9 transcription factor, Collagen type II and Aggrecan) were considerably lower in the rhCII hydrogels than in the high‐density pellets, but at the end of the 84‐day culture period, all the cartilage marker genes analysed were expressed at a similar level. Interestingly, the expression of the matrix metallopeptidases (MMP)‐13, MMP‐14 and MMP‐8, i.e. extracellular collagen network‐degrading enzymes, were transiently upregulated in the rhCII hydrogel, indicating active matrix reorganization. This study demonstrated that the rhCII hydrogel functions as a xeno‐free platform for BM‐MSC chondrogenesis, although the process is delayed. The reversible catabolic reaction evoked by the rhCII hydrogel might be beneficial in graft integration in vivo and pinpoints the need to further explore the use of hydrogels containing recombinant extracellular matrix (ECM) proteins to induce the chondrogenesis of MSCs. Copyright

Chapter 5 – Stem Cell-Based Approaches for Cartilage Tissue Engineering: What Can We Learn From Developmental Biology

Abstract Articular cartilage is an extraordinary tissue with unique physiological and mechanical characteristics. However, when damaged, articular cartilage has a very limited capacity to repair itself. Unfortunately, current clinical interventions are not optimal to restore the native characteristics of this tissue. To overcome this problem, a growing interest is moving towards applying concepts akin to those observed during cartilage development. At the most simple level, limb mesenchymal progenitor cells first aggregate into a condense structure, and following a series of defined steps, they are then specified into all tissues found in the joint, articular cartilage included. However, these developmental stages are meticulously regulated by chemical-physical stimuli capable of inducing the expression of important cartilage-specific transcription factors and ultimately the articular chondrocyte fate. Here we provide an overview about how the continuously growing body of knowledge of cartilage development in utero is driving the design of new stem cell-based strategies for cartilage repair.