Mesenchymal Stem Cells Arrest Intervertebral Disc Degeneration Through Chondrocytic Differentiation and Stimulation of Endogenous Cells  Fan Yang, Victor YL Leung, Keith DK Luk, Danny Chan, Kenneth MC Cheung  Molecular Therapy  Volume 17, Issue 11, Pages 1959-1966 (November 2009) DOI: 10.1038/mt.2009.146 Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Annulus puncture could induce the progressive degeneration of murine intervertebral disc. (a–d) Safranin O staining of the intervertebral discs from pre-op to 12 weeks after the puncture. Progressive degenerative changes were observed at (a) pre-op, (b) 2 weeks, (c) 6 weeks, and (d) 12 weeks after the puncture. Bar = 200 µm (e) the histological score increased from pre-op to 2 weeks (P < 0.05), then increased from 2 to 12 weeks (P < 0.05) after the puncture. (f) Disc height index (%DHI) decreased continuously at 2, 6 weeks (P < 0.05) and 12 weeks (P < 0.01) after the puncture. (g) The expression of Col2a1, Aggrecan, and Sox9 were significantly downregulated from pre-op to 12 weeks after the puncture (P < 0.01). (h) Continuous decrease of GAG/DNA from pre-op to 12 weeks after the puncture. The GAG content decreased significantly at 6 and 12 weeks were compared with the pre-op control group (P < 0.05).GAG, glycosaminoglycan. Molecular Therapy 2009 17, 1959-1966DOI: (10.1038/mt.2009.146) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Bone marrow derived mesenchymal stem cells (BMSCs) isolated from GFP mice. (a,b) BMSC were cultured in Dulbecco's modified Eagle medium at P1. The cells observed in the (a) bright field can emit green fluorescence under (b) fluorescence microscope. Bar = 100 µm. Immunostaining of (c) Stro-1, (d) CD73, (e) CD34, (f) CD45 of the cultured BMSC. BMSC could express Stro-1 and CD73, whereas could not express CD34 and CD45. Bar = 40 µm (c) reverse transcription–PCR analysis of Sox9 and Col2a1 in articular chondrocytes and BMSC. (h) Experimental design for transplantation procedure of BMSC in the degenerated disc. GFP, green fluorescent protein. Molecular Therapy 2009 17, 1959-1966DOI: (10.1038/mt.2009.146) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 The progressive degeneration of murine disc was arrested by BMSC. (a–i) Safranin O staining of intervertebral discs isolated from normal group (a,b,c), degeneration group (d,e,f) and treatment group (g,h,i) at pre-op (a,d,g), 4 weeks (b,e,h) and 24 weeks (c,f,i) after injection. In normal disc (a–c), lamellas in annulus fibrosus (AF) align parallel and nucleus pulposus (NP) is a highly organized structure; degenerative changes were observed in degeneration group (e,f): the lamellas in AF became disorganized, clusters of cells formed in NP at 4 weeks (e) and further decreased at 24 weeks (f); regenerative changes were observed in treatment group (h,i): the lamellas in AF maintained regular at 4 week (h) and 24 week (i), cellular components maintained in NP at 4 weeks (h) and changed into glycosaminoglycan rich phenotype at 24 weeks (i). Bar = 200 µm. (j) Change of DHI in three different groups: At 24 weeks postinjection %DHI in treatment group was significantly higher than the degeneration group (P < 0.05). (k) Histological score of normal, degeneration and treatment group at 4 weeks and 24 weeks after the injection. Histological score in D group was significantly higher than T group at both 4 weeks (P < 0.05) and 24 weeks (P < 0.05). DHI, disc height index. Molecular Therapy 2009 17, 1959-1966DOI: (10.1038/mt.2009.146) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Upregulated ECM gene expression and increased extracellular matrix in the regenerated discs. (a) Gene expression analysis of Col2a1, Aggrecan, and Sox9 in normal, degeneration and treatment group at 24 weeks postinjection. The expression level in treatment group was significantly higher than degeneration group (P < 0.05). (b) Change of GAG contents in different groups: GAG increased significantly in treatment group compared with degeneration group (P < 0.01). (c–k) Different expression pattern of Col2a1 were observed in normal (c,d,e), degeneration (f,g,h) and treatment group (i,j,k) at pre-op (c,f,i), 4 weeks (d,g,j) and 24 weeks (e,h,k) postinjection. Positive signals in degeneration group decreased from 4 weeks (g, arrow) to 24 weeks (h, arrow). Positive signals continuously increased in treatment group from 4 weeks (j, arrow) to 24 weeks (k, arrow). Bar = 200 µm. (l–q) Immunostaining of collagen II of the normal (l,m), degeneration (n,o) or treatment group (p,q) from 4 weeks to 24 weeks. Collagen II accumulated around the cell clusters from 4 to 24 weeks (p,q). Bar = 50 µm. GAG, glycosaminoglycan. Molecular Therapy 2009 17, 1959-1966DOI: (10.1038/mt.2009.146) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 In vivo chondrocytic differentiation of bone marrow derived mesenchymal stem cell (BMSC) in the regenerated discs. (a) Quantification of GFP-positive cells in the murine discs. GFP-positive cells decreased significantly from 4 to 24 weeks (P < 0.05), the number of GFP-positive cells at 4 weeks was set as 100%. (b–e) GFP staining (b,c) and safranin O staining (d,e) of the BMSC. BMSC changed from spindle shape (b,d, arrow) into the round chondrocytic phenotype (c,e, arrow). Bar = 10 µm. (f,g) Double staining of the injected stem cells using GFP antibody (red) with Col2a1 probe (f, green) or Sox9 antibody (g, green) at 4 weeks postinjection. 4′,6-diamidino-2-phenylindole was used to stain the cell nucleus (blue) and the colors were labeled in the image. Bar = 50 µm. (h) Ratio changes of Col2a1 and Sox9 positive cells at 4 weeks and 24 weeks postinjection. The ratio increased significantly from 4 weeks to 24 weeks for Col2a1 (P < 0.05) and Sox9 (P < 0.05).GFP, green fluorescent protein. Molecular Therapy 2009 17, 1959-1966DOI: (10.1038/mt.2009.146) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Endogenous NP cells increased and differentiated into the chondrocytic phenotype. (a–d) GFP staining of the nucleus pulposus in degeneration group (a,c) and treatment group (b,d) at 4 weeks (a,b) and 24 weeks (c,d). The endogenous NP cells which were GFP negative could be observed at both time points (arrow). Bar = 50 µm (e) comparison of the number of endogenous NP cells between normal group and treatment group. The cell number in treatment group was significantly higher than degeneration group at 4 week (P < 0.01) and 24 weeks (P < 0.05). (f–i) Different molecular phenotypes of cells were observed from the double staining, including Col2a1-expressing (f) and nonexpressing (g) GFP-negative endogenous cells as well as Col2a1 expressing (h) and nonexpressing (i) GFP-positive bone marrow derived mesenchymal stem cell. (j) The number of Col2a1 positive endogenous NP cell changes from 4 weeks to 24 weeks. The positive NP cells significantly increased in treatment group compared with degeneration group from 4 to 24 weeks (P < 0.05). GFP, green fluorescent protein; NP, nucleus pulposus. Molecular Therapy 2009 17, 1959-1966DOI: (10.1038/mt.2009.146) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions