Transient vascularization of transplanted human adult-derived progenitors promotes self-organizing cartilage

Abstract

Millions of patients worldwide are affected by craniofacial deformations caused by congenital defects or trauma. Current surgical interventions have limited therapeutic outcomes; therefore, methods that would allow cartilage restoration are of great interest. A number of studies on embryonic limb development have shown that chondrogenesis is initiated by cellular condensation, during which mesenchymal progenitors aggregate and form 3D structures. Here, we demonstrated efficient regeneration of avascular elastic cartilage from in vitro-grown mesenchymal condensation, which recapitulated the early stages of chondrogenesis, including transient vascularization. After transplantation of vascularized condensed progenitors into immunodeficient mice, we used an intravital imaging approach to follow cartilage maturation. We determined that endothelial cells are present inside rudimentary cartilage (mesenchymal condensation) prior to cartilage maturation. Recreation of endothelial interactions in culture enabled a recently identified population of adult elastic cartilage progenitors to generate mesenchymal condensation in a self-driven manner, without requiring the support of exogenous inductive factors or scaffold materials. Moreover, the culture-grown 3D condensed adult-derived progenitors were amenable to storage via simple freezing methods and efficiently reconstructed 3D elastic cartilage upon transplantation. Together, our results indicate that transplantation of endothelialized and condensed progenitors represents a promising approach to realizing a regenerative medicine treatment for craniofacial deformations.

Figure 5. In vitro–grown condensed progenitor cells efficiently reconstruct human elastic cartilage in vivo.

(A) Gross observation of the transplants at days 3, 15, and 30. Conventional pellet culture transplants (dotted black outlines) were placed into the contralateral portion of the vascularized transplants (blue outlines). (B) Alcian blue–stained sections of the vascularized transplants and pellet transplants at day 60 (n = 3 independent transplantation experiments). Scale bars: 200 μm. (C) Quantification of Alcian blue–positive areas for the widest points of the sections. Data represent the mean ± SEM (n = 3, *P < 0.05).

Figure 4. Recapitulation of transient vascularization prior to human cartilage progenitor maturation.

(A) Macroscopic observation of transplanted day-2 self-condensed hCPCs with HUVECs at multiple time points, showing the initial vascularization and subsequent vessel regression. Scale bar: 2 mm. (B) Intravital visualization of the vasculature inside the transplanted tissues along with the surrounding recipient vessels. Green, dextran; red, CPCs; blue, mouse-specific Alexa Fluor 647–conjugated CD31. Scale bars: 500 μm and 75 μm (inset). (C) Time-dependent changes in the number of transplanted HUVECs over CPCs. Upper panels are representative images for the subsequent quantification analysis. Data represent the mean ± SD (n = 3, *P < 0.01). Green, HUVECs; red, CPCs. (D–G) Safranin O (D) and EVG (E) staining of vascularized tissue transplants at day 30 indicated a significant number of cartilage ECM proteins characteristic of elastic cartilage. Aggrecan (green) and collagen I (red) coimmunostaining (F) showed that the reconstructed cartilage developed both perichondral and chondral layers. Human CD31 immunostaining (G) showed that human endothelial cells remained in the perichondral layer, similar to that observed in normal ear cartilage. Scale bars: 100 μm.

Figure 3. Protocol optimization for growing prevascularized condensation from human progenitor cells.

(A) Comparison between Matrigel-precoated and normal cell culture plates on which hCPC single cultures or hCPC and endothelial cell cocultures were plated, showing that Matrigel presolidification was essential to induce the 3D condensation of hCPCs and HUVECs. Scale bar: 2 mm. (B) Confocal imaging of in vitro–generated condensed tissue at day 4. Green indicates HUVECs; red indicates hCPCs. Scale bar: 750 μm. (C) Protocol optimization of the hCPC and HUVEC mixing ratio for the regeneration of human cartilage. See also Supplemental Figure 3. *P < 0.01 by Mann-Whitney U test with Bonferroni’s correction.

Figure 2. Self-driven condensation of hCPCs by endothelial cell coculture on a soft substrate.

(A) Schematic diagram of our culturing method. (B) Time-dependent changes in macroscopic and fluorescence microscopic images in vitro. hCPCs self-assembled into condensed 3D immature cartilage in vitro, without the aid of scaffolds or soluble factors. Green indicates HUVECs; red indicates human cartilage from CPCs. Bottom row original magnification: ×10.

Figure 1. Rudimentary ear cartilage (mesenchymal condensation) is transiently vascularized in vivo.

(A) Gross observations of E18.5 CAG-EGFP murine immature cartilage transplants at multiple time points. (B) Immunohistological analyses of mCD31 and laminins in ear cartilage from P0 to P30. Dotted line indicates the border between the chondral and perichondral layers of the mouse ear cartilage. Scale bar: 50 μm. (C) Intravital confocal images of the same field of view at 3, 7, and 10 days after transplantation. Arrowhead indicates the vascularized area of the transplants. Endothelial cells and blood perfusion were visualized intravitally using Alexa Fluor 647–conjugated mCD31 antibody and fluorophore-conjugated dextran injected via the tail vein. Scale bars: 75 μm. (D) Formation of mature cartilage 20 days after transplantation. The developed chondral layer was composed of homogenous polygonal mature chondrocytes, whereas the perichondrium (double-headed arrow, outer fibrous layer) was composed of spindle-shaped putative progenitor cells that were proximate to blood vessels, similar to normal ear cartilage. Scale bars: 200 μm (top and middle panels) and 25 μm (bottom panel). (E) Terminal differentiation from murine progenitor cells into mature chondrocytes after transplantation into a cranial window. Alcian blue and EVG staining confirmed the formation of mature elastic cartilage from E18.5 immature cartilage transplants after 20 days compared with E18.5 and P30 cartilage sections. Tx; transplantation. Scale bars: 100 μm.