Bone Marrow Mesenchymal Stem Cells Spontaneously Make Cartilage After Blockage of VEGF Signaling


Bone marrow-derived mesenchymal stem cells (MSCs) can be induced to make cartilage by incubating the cells with particular growth factors.  Unfortunately, batches of MSCs show respectable variability from patient-to-patient.  Therefore the growth factor-dependent method suffers from poor efficacy, limited reproducibility from batch-to-batch, and the cell types that are induced are not always terribly stable.  Finding a better way to make cartilage would certainly be a welcome addition to regenerative treatments,

Cartilage that coats the ends of bones is known as articulate cartilage, and articular cartilage lacks blood vessels.  Therefore, is it possible that inhibiting blood vessel formation could conveniently push MSCs to differentiate into cartilage-making chondrocytes?

A new paper by Ivan Martin and Andrea Basil from the University Hospital Basel and their colleagues have used this very strategy to induce cartilage formation in MSCs from bone marrow.

Martin and others isolated MSCs from bone marrow aspirates from human donors.  These cultured human MSCs were then genetically engineered with modified viruses to express a receptor for soluble vascular endothelial growth factor (VEGF) that binds this growth factor, but fails to induce any intracellular signals.  Such a receptor that binds the growth factor but does not induce any biological effects is called a “decoy receptor,” and decoy receptors efficiently sequester or vacuum up all the endogenous VEGF.  VEGF is the major blood vessel-inducing growth factor and it is heavily expressed during development, by cancer cells, and during healing.

After expressing the decoy VEGF receptor in these human MSCs, these genetically engineered cells were grown on collagen sponges and then implanted in immunodeficient mice.  If the implanted MSCs were not genetically engineered to express decoy VEGF receptors, they induced for formation of vascularized fibrous tissue.  However, the implantation of genetically engineered MSCs that expressed the decoy VEGF receptor efficiently and reproducibly differentiated into chondrocytes and formed hyaline cartilage. This is significant because headline cartilage is the very type of cartilage found at articular surfaces where the ends of bones come together to form joints.

In vivo chondrogenesis. Histological staining with Safranin-O for glycosaminoglycans and immunohistochemistry for type II collagen of engineered tissue generated by naïve (control) or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Fluorescence staining with DAPI (in blue) and a specific anti-human nuclei antibody (in red) of constructs generated by control or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MSC, bone marrow-derived mesenchymal stromal/stem cell.
In vivo chondrogenesis. Histological staining with Safranin-O for glycosaminoglycans and immunohistochemistry for type II collagen of engineered tissue generated by naïve (control) or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Fluorescence staining with DAPI (in blue) and a specific anti-human nuclei antibody (in red) of constructs generated by control or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MSC, bone marrow-derived mesenchymal stromal/stem cell.

This articular cartilage was quite stable and showed no signs of undergoing the chondrocytes enlargement found in terminally differentiated cartilage that is ready to form bone.  This stability was maintained for up to 12 weeks.

In vivo cartilage stability. Immunohistochemistry for type X collagen, BSP, and MMP-13 on sections of hypertrophic cartilage generated in vitro by MSCs (as a positive control) and on sections of the cartilaginous constructs generated in vivo by sFlk1 MSCs 12 weeks after implantation. Scale bar = 50 µm. Abbreviations: BSP, bone sialoprotein; MMP-13, metalloproteinase-13; MSC, bone marrow-derived mesenchymal stromal/stem cell.
In vivo cartilage stability. Immunohistochemistry for type X collagen, BSP, and MMP-13 on sections of hypertrophic cartilage generated in vitro by MSCs (as a positive control) and on sections of the cartilaginous constructs generated in vivo by sFlk1 MSCs 12 weeks after implantation. Scale bar = 50 µm. Abbreviations: BSP, bone sialoprotein; MMP-13, metalloproteinase-13; MSC, bone marrow-derived mesenchymal stromal/stem cell.

Why did inhibition of VEGF signaling induce cartilage?  Inhibition of angiogenesis induced low oxygen tensions, which activated a growth factor called transforming growth factor-β.  Activation of the TGF-beta pathway robustly enhanced the formation of articular cartilage.

In vitro chondrogenesis at different oxygen tensions. Histological staining with Safranin-O and immunohistochemistry for type II collagen on constructs generated in vitro by naïve MSC cultured with (A) or without (B) TGFβ3 supplementation at 2% or 20% of oxygen tension. Scale bar = 50 µm. Expression levels of the mRNA for type II and X collagen, Gremlin-1, IHH TGFβ1 were quantified in pellets generated by naïve bone marrow-derived mesenchymal stromal/stem cells (C, D) cultured in the two different oxygen tensions. ∆Ct values were normalized to expression of the GAPDH housekeeping gene, and results are shown as mean ± SD (n = 6 samples/group from 3 independent experiments). ∗, p < .05, ∗∗∗, p < .001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHH, Indian hedgehog; TGFβ, transforming grown factor-β.
In vitro chondrogenesis at different oxygen tensions. Histological staining with Safranin-O and immunohistochemistry for type II collagen on constructs generated in vitro by naïve MSC cultured with (A) or without (B) TGFβ3 supplementation at 2% or 20% of oxygen tension. Scale bar = 50 µm. Expression levels of the mRNA for type II and X collagen, Gremlin-1, IHH TGFβ1 were quantified in pellets generated by naïve bone marrow-derived mesenchymal stromal/stem cells (C, D) cultured in the two different oxygen tensions. ∆Ct values were normalized to expression of the GAPDH housekeeping gene, and results are shown as mean ± SD (n = 6 samples/group from 3 independent experiments). ∗, p < .05, ∗∗∗, p < .001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHH, Indian hedgehog; TGFβ, transforming grown factor-β.

Cartilage formation from MSCs was induced by blocking VEGF-mediated angiogenesis.  These results represent a remarkable advance in cartilage formation that can be used for regenerative treatments.  This cartilage formation was spontaneous and efficient and if it can be carried out with VEGF-inhibiting drugs rather than genetic engineering techniques, then we might have a transferable technique for making cartilage in the laboratory to treat osteoarthritis and other joint-based maladies.  Clinical trials will be required, but this is certainly an auspicious start.

Cultured Skin-Based Stem Cells Regenerate Hair Follicles and Sebaceous Glands


It has been over ten years since the development of cultured skin substitutes or CSSs. CSSs consist of cultured epidermis from the patients and dermal fibroblasts that can form a good epidermal layer. However, CSSs do not form hair follicles or sebaceous glands which makes for mighty dry skin. Therefore, it is highly preferable to make at skin substitute that can form such epidermal appendages.

Fortunately the skin possesses several types of stem cells for use in regenerative medicine. Epidermal stem cells (Epi-SCs) in the basal layer of the epidermis that constantly provide new cells to the epidermis (the uppermost layer of the skin). Unfortunately, adult Epi-SCs cannot form hair follicles, but they can if they are combined with embryonic or newborn dermal cells. Dermal papilla (DP) cells can induce hair follicles, but the availability of DP cells has greatly limited work with them. Adult dermal skin also contains multipotent SKPs or skin-derived precursors. Injection of SKPs underneath the skin of mice, leads to the induction of new hair follicles (Biernaskie et al., 2009, Cell Stem Cell 5:610-623). This suggests that SKPs might be applicable in a clinical setting to induce hair follicles in cultured skin substitutes.

A new paper by Yaojiong Wu and coworker from the Shenzhen Key Laboratory of Health Sciences and Technology, in collaboration with Edward E. Tredget from University College Dublin has successfully induced the growth of hair follicles and sebaceous glands in a cultured skin substitute. They combined cultured Epi-SCs and SKPs, from mice and humans, and embedded them into a hydrogel. These stem cell-embedded hydrogels were then implanted into immunodeficient mice with substantial skin wounds.

The results were remarkable. The implants able to induce the formation of new epidermis with hair follicles. Furthermore, when Epi-SCs and SKPs taken from human scalps were used in these experiments, they worked just as well as those taken from mice.

Hair neogenesis with cultured epidermal stem cells (Epi-SCs) and skin-derived precursors (SKPs). (A): Putative epidermal stem cells residing in the basal layer of neonatal mouse epidermis expressed CD49f (red) in immunofluorescence stain, and mature keratinocytes in the top layers of the epidermis expressed cytokeratin (CK)6 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole. (B–E): Cultured Epi-SCs derived from neonatal mice (B) were positive for CD49f (C) and CK15 (D) in immunofluorescence stain; fluorescence-activated cell sorting analysis of the Epi-SCs indicated high levels of surface CD29 and CD49f (E). (F): The expression level of CD49f decreased progressively upon successive passages (P) in culture as determined by immunofluorescence analysis (in relation to the fluorescence intensity of P0 cells). Triple wells were used for each of the above experiments, and each experiment was repeated three times with similar results (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (G): Hair genesis of cultured Epi-SCs in different passages. Cultured Epi-SCs derived from neonatal mice in different passages (P0 to P5) were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal dermal cells (fresh D) in Matrigel; dermal cells alone or freshly isolated neonatal epidermal cells plus dermal cells (fresh E+D) were used as controls. Hair shafts generated 20 days posttransplant were counted (n = 6; ∗, p < .05; ∗∗∗, p < .001). (H–J): SKPs derived from neonatal mice in spheroid culture (H) expressed nestin, fibronectin (I), and BMP6 (J) in immunofluorescence analysis. (K): Hair genesis of SKPs in different passages. SKPs in P0 to P5 were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal mouse epidermal cells (fresh E), and freshly isolated neonatal mouse epidermal cells alone or in combination with freshly isolated neonatal mouse dermal cells (fresh E+D) were used as controls. Twenty days posttransplant, hairs generated were counted (n = 6; ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (L-N): Cultured Epi-SCs and SKPs in hair genesis. Combinations of cultured neonatal mouse Epi-SCs (P0 to P3) and SKPs (P0 to P3) were engrafted into excisional wounds in nude mice, and the number of hairs generated were counted 20 days posttransplant (n = 3, ∗, p < .05). (L). A representative image of hairs generated 20 days after a transplantation of P1 Epi-SCs and SKPs (M). Immunofluorescence analysis of the skin tissue with hair genesis showed densely populated hair follicles and sebaceous glands (N). Scale bars = 50 μm. Abbreviations: BM, basement membrane; BMP6, bone morphogenetic protein 6; CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; Derm, dermis; Epi, epidermis; Epi-SC, epidermal stem cells; FITC, fluorescein isothiocyanate; fresh D, freshly isolated neonatal dermal cells; fresh D+E, freshly isolated neonatal epidermal cells plus dermal cells; HF, hair follicle; HS, hair shafts; P, passage; PE, phycoerythrin.
Hair neogenesis with cultured epidermal stem cells (Epi-SCs) and skin-derived precursors (SKPs). (A): Putative epidermal stem cells residing in the basal layer of neonatal mouse epidermis expressed CD49f (red) in immunofluorescence stain, and mature keratinocytes in the top layers of the epidermis expressed cytokeratin (CK)6 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole. (B–E): Cultured Epi-SCs derived from neonatal mice (B) were positive for CD49f (C) and CK15 (D) in immunofluorescence stain; fluorescence-activated cell sorting analysis of the Epi-SCs indicated high levels of surface CD29 and CD49f (E). (F): The expression level of CD49f decreased progressively upon successive passages (P) in culture as determined by immunofluorescence analysis (in relation to the fluorescence intensity of P0 cells). Triple wells were used for each of the above experiments, and each experiment was repeated three times with similar results (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (G): Hair genesis of cultured Epi-SCs in different passages. Cultured Epi-SCs derived from neonatal mice in different passages (P0 to P5) were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal dermal cells (fresh D) in Matrigel; dermal cells alone or freshly isolated neonatal epidermal cells plus dermal cells (fresh E+D) were used as controls. Hair shafts generated 20 days posttransplant were counted (n = 6; ∗, p < .05; ∗∗∗, p < .001). (H–J): SKPs derived from neonatal mice in spheroid culture (H) expressed nestin, fibronectin (I), and BMP6 (J) in immunofluorescence analysis. (K): Hair genesis of SKPs in different passages. SKPs in P0 to P5 were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal mouse epidermal cells (fresh E), and freshly isolated neonatal mouse epidermal cells alone or in combination with freshly isolated neonatal mouse dermal cells (fresh E+D) were used as controls. Twenty days posttransplant, hairs generated were counted (n = 6; ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (L-N): Cultured Epi-SCs and SKPs in hair genesis. Combinations of cultured neonatal mouse Epi-SCs (P0 to P3) and SKPs (P0 to P3) were engrafted into excisional wounds in nude mice, and the number of hairs generated were counted 20 days posttransplant (n = 3, ∗, p < .05). (L). A representative image of hairs generated 20 days after a transplantation of P1 Epi-SCs and SKPs (M). Immunofluorescence analysis of the skin tissue with hair genesis showed densely populated hair follicles and sebaceous glands (N). Scale bars = 50 μm. Abbreviations: BM, basement membrane; BMP6, bone morphogenetic protein 6; CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; Derm, dermis; Epi, epidermis; Epi-SC, epidermal stem cells; FITC, fluorescein isothiocyanate; fresh D, freshly isolated neonatal dermal cells; fresh D+E, freshly isolated neonatal epidermal cells plus dermal cells; HF, hair follicle; HS, hair shafts; P, passage; PE, phycoerythrin.

Additionally, when the ability of Epi-SCs to differentiate into sebaceous glands was examined, Wu and others showed that Epi-SCs can form the precursors to sebaceous glands, sebocytes. Additionally, the oils secreted by these Epi-SC-derived sebocytes were chemically similar to sebaceous glands from native skin.

Thus, a combination of Epi-SCs and SKPs from human or mouse skin were sufficient to generate newly formed hair follicles and functional sebaceous glands. These results provide knowledge that is potentially transferable to clinical applications for regenerating damaged skin.