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The capacity of digit tip regeneration observed both in rodents and humans establishes a foundation for promoting robust regeneration in mammals. However, stimulating regeneration at more proximal levels, such as the middle phalanges (P2) of the adult mouse, remains challenging. Having shown the effectiveness of transplantation of limb progenitor cells in stimulating limb regeneration in Xenopus, we are now applying the cell transplantation approach to the adult mouse. Here we report that both embryonic and induced pluripotent stem cell (iPSC)-derived limb progenitor-like cells can promote adult mouse P2 regeneration. We have established a simple and efficient protocol for deriving limb progenitor-like cells from mouse iPSCs. iPSCs are cultured as three-dimensional fibrin bodies, followed by treatment with combinations of Fgf8, CHIR99021, Purmorphamine and SB43542 during differentiation. These iPSC-derived limb progenitor-like cells resemble embryonic limbmesenchyme cells in their expression of limb-related genes. After transplantation, the limb progenitor-like cells can promote adult mouse P2 regeneration, as embryonic limb bud cells do. Our results provide a basis for further developing progenitor cell-based approaches for improving regeneration in the adult mouse limbs.
Figure 1. Optimization of cell transplantation in adult mouse D3P2 stump. (a) An amputated P2 digit was covered with a Hyphecan cap immediately after amputation to facilitate healing, and cell transplantation was performed at 12âdpa, by placing cells in fibrin gel under the re-opened wound epithelium. (b) Long-term retention of transplanted cells, observed at 12 weeks post amputation (wpa), with an elongated P2, in contrast to the blunt stump in control P2 amputations. White lines in (b) indicate amputation levels. Scale bars: 1âmm. (câg) Selection of growth factors for cell transplantation in adult D3P2. (c) Distance of cells migrating out from fibrin patch in the presence of growth factors, measured in 10-day cultures. (d) Cross-sections of D3P2 stumps (3 dpt) with cell transplantation alone, or transplanted with cells and growth factor combination BFTW (Bmp2+Fgf8+Thymosin b4+ Wnt3a). The cell transplant is outlined with white dotted lines. (e) Analysis of cell proliferation in D3P2 stumps, at 10âdpt. White arrows indicate examples of EdU+ GFP+ cells. (f) Statistical analysis of GFP+ cells, and EdU+/GFP+ (proliferating) cells in the transplants, **P<0.01, Studentâs t-test, n=3. (g) Hematoxylin and eosin staining on parasagittal sections of D3P2 after transplantation, at 10âdpt, showing larger cell mass in the P2 after transplantation of cells together with growth factors. Black arrowheads indicate amputation levels.
Figure 2. D3P2 regeneration after transplantation of embryonic limb progenitor cells together with growth factors. (a) Examples of adult mouse D3P2 after nGFP embryonic limb progenitor cell transplantation (with BFTW factors), under fluorescent microscope, after skin and soft tissue removal, and by X-ray imaging. GFP+ cells are in the bone regenerate. The green square area is auto fluorescence. X-ray image obtained at 20 weeks post amputation (wpa) is shown. Arrowheads indicate amputation levels. r indicates the regenerated bone. (b) Example of D3P2 transplanted with limb progenitor cells alone. (c) Example of non-regenerating bone in D3P2 implanted with BFTW beads only. Minimal regenerated bone can be detected with OPN (red). (d) Regeneration of bone as measured on X-ray images (determined as r in d). Error bars: standard error. Sizes of samples are shown in parenthesis. P<0.01, analysis of variance (ANOVA; one way) analysis. (e) Restoration of D3P2 determined by r/(length of amputated P2)Ã100%. (fâh) Distribution of transplanted limb progenitor cells in the P2 regenerates. (f) The regenerated bone contains both GFP+ and GFPâ cells. White arrows indicate green cells at the outside surface of the regenerated bone. Red arrows indicate examples of host cells. OPN marks the regenerated bone. (g) GFP+ cell clusters found in the loose connective tissues in the distal stumps, underneath the skin (outlined with dotted line). White arrowheads indicate amputation levels. Scale bars in (a,
b): 0.2âmm; (f, g): 50âμm.
Figure 3. Derivation of limb progenitor-like cells from 3D FB culture of Prx1Cre:mT/mG iPSCs. (a) Diagram of the protocol used in this study. LIF (leukemia inhibitory factor), Chir (CHIR99021, 3âμM), Fgf8 (10âng/ml), Pur (Purmorphamine, 4âμM), SB (SB431542, 2âμM). (b) Examples of FBs freshly made and cultured for 14 days (d14). Inset in d0 is a side view of the hemispherical FB immediately after transfer to culture medium. Multiple budding structures are GFP+ in a d14 FB (b). (c) A smaller bud-like structure, with a GFP+ core covered by tdTomato+ outside layer, revealed in cross-section. (d) Flow cytometry analysis of GFP+ cells in D14 FBs, showing about 40% cells were induced to switch on GFP expression. (e) Real-time PCR detection of gene expression in FBs, for pluripotency markers (Nanog, Oct4, Sox2), lateral plate mesoderm markers (Gata4, Kdr, Meox1) and limb field related genes (Prx1, Gli3, Pitx1, Tbx5). Gene expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and compared with d0 specimens. Results were from three independent experiments. (f) Detection of mesoderm markers Kdr and Pdgfrα at d7 of differentiation. (g) Detection of Fgf10 in day 10 FB cultures. (h) Induced GFP+ cells expressed mesodermal marker α-SMA (in purple), detected by immunofluorescence. (i) Detection of Isl1, Gli3, Tbx5, Pitx1 and Tbx4 in induced GFP+ cells by immunofluorescence. Scale bars: (b): 0.5âmm, (c): 200âμm, (fâi): 20âμm.
Figure 4. Gene expression analysis of induced limb progenitor cells in comparison with embryonic limb bud. (a, b) Hierarchical clustering of Hox genes (a) and transcription factors related to embryonic limb development (b) in FBs, E10.5 fore- and hind-limb, control FB without differentiation (kept in MES-Lif), and 2c-iPSCs. (c) Expression levels of selected genes involved in the determination and development of embryonic limb, showing that these genes were induced in differentiated FBs, but not in control or iPSCs.
Figure 5. Stimulated adult mouse D3P2 regeneration after transplantation of iPSC-derived limb progenitor-like cells. (a) Examples of adult mouse D3P2 with or without limb progenitor-like cell +BFTW transplantation), by X-ray imaging. (b) Statistical analysis of length of bone regeneration and P2 restoration in adult D3P2 with graft (limb progenitor-like cells+BFTW) or without (control). Error bars: standard error, N=9. **P<0.01, Studentâs t-test. (câe) Sections of D3P2 with Trichrome staining, in control (d) or cells+BFTW group (d, e), 6 wpt. Black arrows indicate amputation levels. White dotted boxes indicate areas shown in (e). (e) An enlarged view of the P2 regenerate, showing well-integrated outgrowth of the P2 bone, and marrow formed in the regenerated bone (red asterisk). (f, g) Contribution of GFP+ iPSC-derived limb progenitor cells (exemplified by the white arrow) and GFPâ host cells (exemplified by the red arrows) in the bone regenerate (marked by OPN expression, in red). *Indicates areas of GFP+ cells outside the bone. (h) Contribution of GFP+ iPSC-derived limb progenitor cells in the connective tissues of the adult D3P2. Scale bars: 0.5âmm in (a, c, d), 0.2âmm in (e), 50âμm in (g,
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