Tracking mesenchymal stem cell contributions to regeneration in an immunocompetent cartilage regeneration model
Daniela Zwolanek, … , Thomas Rülicke, Reinhold G. Erben
Daniela Zwolanek, … , Thomas Rülicke, Reinhold G. Erben
Published October 19, 2017
Citation Information: JCI Insight. 2017;2(20):e87322. https://doi.org/10.1172/jci.insight.87322.
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Technical Advance Stem cells Transplantation

Tracking mesenchymal stem cell contributions to regeneration in an immunocompetent cartilage regeneration model

Abstract

It is currently controversially discussed whether mesenchymal stem cells (MSC) facilitate cartilage regeneration in vivo by a progenitor- or a nonprogenitor-mediated mechanism. Here, we describe a potentially novel unbiased in vivo cell tracking system based on transgenic donor and corresponding immunocompetent marker–tolerant recipient mouse and rat lines in inbred genetic backgrounds. Tolerance of recipients was achieved by transgenic expression of an immunologically neutral but physicochemically distinguishable variant of the marker human placental alkaline phosphatase (ALPP). In this dual transgenic system, donor lines ubiquitously express WT, heat-resistant ALPP protein, whereas recipient lines express a heat-labile ALPP mutant (ALPPE451G) resulting from a single amino acid substitution. Tolerance of recipient lines to ALPP-expressing cells and tissues was verified by skin transplantation. Using this model, we show that intraarticularly injected MSC contribute to regeneration of articular cartilage in full-thickness cartilage defects mainly via a nonprogenitor-mediated mechanism.

Authors

Daniela Zwolanek, María Satué, Verena Proell, José R. Godoy, Kathrin I. Odörfer, Magdalena Flicker, Sigrid C. Hoffmann, Thomas Rülicke, Reinhold G. Erben

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Figure 3

Intraarticularly injected MSC contribute to cartilage regeneration in a rat model of full-thickness cartilage defects.

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Intraarticularly injected MSC contribute to cartilage regeneration in a ...
(A) Histochemical ALPP staining of the distal femoral joint surface with a full-thickness cartilage defect in the patellar groove 1 and 28 days after intraarticular injection of serum or 1 × 107 MSC from Tg(ALPP) donor rats. Arrows show ALPP-positive staining in the defects. ALPP staining was absent in WT or Tg(ALPPm) recipients injected with MSC from Tg(ALPP) donors or serum, respectively. Scale bar: 250 μm. (B) Quantification of the ALPP-positive area within the full-thickness defects. Dots represent ALPP-positive area per animal. n ≥ 5 per group. *P < 0.05, ALPP → ALPPm recipients vs. all other groups by Kruskal-Wallis test followed by Mann-Whitney U test. (C) Histochemical ALPP staining of cryosections from femoral full-thickness cartilage defects 1 and 28 days after injection of 1 × 107 MSC from Tg(ALPP) donor rats into the knee of Tg(ALPPm) recipients. Insets show ALPP-positive cells at the bottom of the defects. Scale bar: 50 μm. n = 3 animals per group. (D) Toluidine blue staining of cryosections from full-thickness cartilage defects, 28 days after injection of 1 × 107 MSC from Tg(ALPP) donor rats into WT or Tg(ALPPm) recipients. Black arrows indicate induction of neocartilage formation within the defects of Tg(ALPPm) animals. Scale bar: 50 μm. n = 3 per group. (E, G, and H) Immunofluorescence staining of cryosections from full-thickness cartilage defects using anti–collagen II (anti-COL2, green) and anti-ALPP antibodies (red) (E), anti-ALPP (green) and anti-Sox9 antibodies (red) (G), or anti-COMP (green) and anti-ALPP antibodies (red) (H) 1 month after injection of 1 × 107 MSC from Tg(ALPP) donor rats into WT or Tg(ALPPm) recipients. White arrows in insets show ALPP-expressing cells at the bottom of the defect surrounded by COL2-containing matrix (E) and ALPP- and SOX9-coexpressing cells within the defect (G) in Tg(ALPPm) recipients. Neither ALPP-expressing cells, new COL2 matrix, nor ALPP- SOX9-coexpressing cells were found in WT recipients. Scale bar = 50 μm. n = 3 per group. (F) Quantification of the COL2-stained area within the defects in cryosections 28 days after injection of MSC from Tg(ALPP) donors in WT or Tg(ALPPm) recipients. Dots represent means of COL2-stained area per animal. n = 3 per group. *P < 0.05 by Student’s t test. ALPP, human placental alkaline phosphatase; ALPPm, ALPPE451G mutant; ALPP → ALPPm, transplantation from Tg(ALPP) donor into Tg(ALPPm) recipient; TX, transplantation.