SCA-1/Ly6A Mesodermal Skeletal Progenitor Subpopulations Reveal Differential Commitment of Early Limb Bud Cells

Abstract

At early developmental stages, limb bud mesodermal undifferentiated cells are morphologically indistinguishable. Although the identification of several mesodermal skeletal progenitor cell populations has been recognized, in advanced stages of limb development here we identified and characterized the differentiation hierarchy of two new early limb bud subpopulations of skeletal progenitors defined by the differential expression of the SCA-1 marker. Based on tissue localization of the mesenchymal stromal cell-associated markers (MSC-am) CD29, Sca-1, CD44, CD105, CD90, and CD73, we identified, by multiparametric analysis, the presence of cell subpopulations in the limb bud capable of responding to inductive signals differentially, namely, sSca⁺ and sSca^- cells. In concordance with its gene expression profile, cell cultures of the sSca⁺ subpopulation showed higher osteogenic but lower chondrogenic capacity than those of sSca^-. Interestingly, under high-density conditions, fibroblast-like cells in the sSca⁺ subpopulation were abundant. Gain-of-function employing micromass cultures and the recombinant limb assay showed that SCA-1 expression promoted tenogenic differentiation, whereas chondrogenesis is delayed. This model represents a system to determine cell differentiation and morphogenesis of different cell subpopulations in similar conditions like in vivo. Our results suggest that the limb bud is composed of a heterogeneous population of progenitors that respond differently to local differentiation inductive signals in the early stages of development, where SCA-1 expression may play a permissive role during cell fate.

Keywords: SCA-1/Ly6A; chondrogenesis; limb bud; progenitor cell; recombinant limbs; tenogenic differentiation.

FIGURE 1

In vivo detection of two MSP limb bud subpopulations based on MSC-am localization. (A) Transversal section of an E10.5 mouse embryo to show the histological organization at this stage; the immunodetections of CD29, SCA-1, CD44, CD105, CD90, and CD73 markers in E10.5 limb buds are shown. The limb bud for each MSC-am marker corresponds to the region inside the square in panel (A). (B) Dot plots of the multiparametric flow cytometry analysis showing the hierarchy to identify sSca⁺ and sSca^– subpopulations in E10.5, E11.5, and E12.5 mouse limbs. The number of positive cells for each subpopulation is expressed in percentage; ±mean standard deviation. (C) Comparison of the expression percentage of the sSca⁺ subpopulation between E10.5, E11.5, and E12.5 limbs. Statistical significance was set as follows: E10.5 vs. E11.5 p > 0.0061; 10.5 vs. 12.5 p > 0.0023; E11.5 vs. E12.5 p > 0.4079, **p < 0.005. NT, neural tube; AGM, aorta-gonad-mesonephros; Do, dorsal; V, ventral; Pr, proximal; D, distal. In (A) AER is marked with an asterisk. The scale bar is set at 200 μm and is representative for all images.

FIGURE 2

sSca⁺ and sSca^– limb bud subpopulations are differentially committed to limb lineages. (A) In vitro osteogenic (alizarin red staining) and (B) chondrogenic (Alcian blue staining) differentiation capacity of freshly isolated sSca⁺ and sSca^– subpopulations compared to total cells. Inset (b) corresponds to magnification showed in b′. Comparison of the expression profile by qRT-PCR of undifferentiated zone genes (C) and master differentiation genes (D) between sSca⁺, sSca^– subpopulations and total cells. Data represent three independent experiments. Statistical significance was set as follows: ***p < 0.0001, **p < 0.005, *p < 0.05. Scale bar set is at 1 mm for all images in panels (A,B), for b′ is set at 100 μm.

FIGURE 3

Sca-1 expression is sufficient to induce Scx and inhibits chondrogenesis. Three days of micromass cultures of non-electroporated (NE), sham, and Sca-1 electroporated limb bud cells (eSca-1⁺). (A) Chondrogenic phenotype by Alcian blue staining and (B) Nodule quantitation are shown. (C) In situ hybridization of Sox9 and Scx. Expression profile by qRT-PCR of master differentiation genes of chondrogenic, tenogenic, and osteogenic lineages (D) and myogenic genes (E) in eSca-1⁺ cells. Expression relative to sham cells is shown (set to 1.0, dashed red line). Data represent at least three independent experiments. Statistical significance represents ***p < 0.0005, **p < 0.005, *p < 0.05. Scale bar is set at 2 mm and is representative for all images.

FIGURE 4

The morphogenetic capacity of Sca-1 limb bud cells in the recombinant limb model. (A) Schematic representation of the experimental design of recombinant limbs experiments. Recombinant limbs (RLs) were performed with non-electroporated (NE), sham (GFP), and Sca-1 electroporated limb bud cells (e-Sca-1⁺). Mesodermal hindlimb cells and ectoderms were obtained from 22HH embryos. Electroporated (GFP or e-Sca-1) and NE recombinant limbs were implanted in 22HH chick embryos and collected after 2 (a–f) or 6 (g–l) days. (B) Alcian blue stain to evidence skeletal elements (a,c,e,g,i,k). Sagittal slices of RL after Alcian blue staining stained with hematoxylin and eosin (b,d,f,h,j,l). Square delimits the magnification for b′,d′,f′. Scale bars are representative for all conditions representing 100 μm. qRT-PCR of chondrogenic (C), tenogenic (D), and tendon cell fate regulating genes (E) of the e-Sca⁺ 48h RLs relative to the GFP-RLs is shown (set to 1.0, dashed red line). GFP-RLs and NE-RLs showed no statistical differences between all genes (data no show). Data represent three independent experiments. Statistical significance was set as follows: **p < 0.005, *p < 0.05.