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.

Trajectory-centric Framework TrajAtlas reveals multi-scale differentiation heterogeneity among cells, genes, and gene module in osteogenesis

“…Besides, we found an MSC-like cell state that highly expressed adult stem cell maker Ly6a (stem cell antigen-6) (Supplementary figure 5e). This population was notably present during the fetal stage in the head and limb regions (Figure 2d), as described in previous studies 34 38 . Consequently, we termed this cluster Ly6a + MSCs 38 .…”

“…This population was notably present during the fetal stage in the head and limb regions (Figure 2d), as described in previous studies 34 38 . Consequently, we termed this cluster Ly6a + MSCs 38 .…”

Recombinant Limb Assay as in Vivo Organoid Model

“…Cells from the AER express Fgf8 , which along with Wnt3a , maintains the mesodermal cells underneath the AER in an undifferentiated, proliferative state ( Ohuchi et al, 1997 ; ten Berge et al, 2008 ). As the limb bud grows, the undifferentiated cells underneath the AER begin to differentiate toward the chondrogenic and tenogenic lineages when they stop receiving signals from the AER ( Dollé et al, 1989 ; Roselló-Díez et al, 2014 ; Marín-Llera et al, 2019 ; Marin-Llera et al, 2021 ). The zone of polarizing activity (ZPA) controls limb bud AP polarity and is located at the posterior margin of the limb bud.…”

“…Based on these characteristics the RL model is a well-suited to evaluate the biology of stem and progenitor cells isolated directly from an organism (e.g., LBM cells). The RL system’s versatility permits the creation of multiple combinations of cells from different sources, developmental stages, or positions along the limb, whole (undissociated), or reaggregated cells, even until modified LBM cells overexpress distinct molecules, as shown by Marín-Llera et al, 2021 .…”

“…Notably, the embryonic signals provided by the ectoderm induce gene expression in a spatiotemporal manner driving the 3D organization of a limb-like structure by recapitulating the developmental programs that occur during limb development. Although this model has been mainly used to understand chicken limb development, different approaches have also been reported, including interspecies grafting ( Fernandez-Teran et al, 1999 ), the use of different combinations of mutant and wild-type mesoderm or ectoderm ( Kuhlman and Niswander, 1997 ), or limb mesodermal cells modified by electroporation ( Marín-Llera et al, 2021 ). From these, it is evident that the RL experimental model is adaptable to diverse scenarios.…”