The enhancer landscape predetermines the skeletal regeneration capacity of stromal cells

Abstract

Multipotent stromal cells are considered attractive sources for cell therapy and tissue engineering. Despite numerous experimental and clinical studies, broad application of stromal cell therapeutics is not yet emerging. A major challenge is the functional diversity of available cell sources. Here, we investigated the regenerative potential of clinically relevant human stromal cells from bone marrow (BMSCs), white adipose tissue, and umbilical cord compared with mature chondrocytes and skin fibroblasts in vitro and in vivo. Although all stromal cell types could express transcription factors related to endochondral ossification, only BMSCs formed cartilage discs in vitro that fully regenerated critical-size femoral defects after transplantation into mice. We identified cell type–specific epigenetic landscapes as the underlying molecular mechanism controlling transcriptional stromal differentiation networks. Binding sites of commonly expressed transcription factors in the enhancer and promoter regions of ossification-related genes, including Runt and bZIP families, were accessible only in BMSCs but not in extraskeletal stromal cells. This suggests an epigenetically predetermined differentiation potential depending on cell origin that allows common transcription factors to trigger distinct organ-specific transcriptional programs, facilitating forward selection of regeneration-competent cell sources. Last, we demonstrate that viable human BMSCs initiated defect healing through the secretion of osteopontin and contributed to transient mineralized bone hard callus formation after transplantation into immunodeficient mice, which was eventually replaced by murine recipient bone during final tissue remodeling.

Batch Effects during Human Bone Marrow Stromal Cell Propagation Prevail Donor Variation and Culture Duration: Impact on Genotype, Phenotype and Function

“…We conducted quality control using FASTQC and removed residual adapter sequences and low-quality reads using Trimmomatic. We mapped reads to the Ensembl GRCh38 human genome using Bowtie 2 as described previously [9]. We submitted raw reads to the GEO database (NUMBER: GSE194303).…”

“…Chondrogenic differentiation was performed in 3D format as previously published with minor modification [7,9]. Briefly, 0.5 × 10 6 BMSCs were seeded at indicated time points after culture onto rat collagen I-coated (C3867, Sigma Aldrich, Burlington, MA, USA) transwell inserts (3470-clear, Corning, New York, NY, USA).…”

“…Disks were harvested, weighed after removing remaining culture medium thoroughly and subsequently formalin-fixed, paraffin-embedded and processed into 4 µm sections. Immunohistochemistry and Bern scoring [27] were done exactly as described previously [9].…”

“…To validate skeletal regenerative function of the different early and late cell products we selected organoid-like 3D cartilage disk formation as one of the most stringent readouts for endochondral differentiation [7,9]. All hPL-and three of four FBS-driven P1 cell products displayed competence to form 3D cartilage discs under appropriate culture conditions.…”

“…BMSC-derived scaffold-free cartilage organoid-like disk transplants could completely regenerate critically sized femur defects in a humanized mouse model. The skeletal regeneration competence was found to be predetermined by a discerning epigenetic landscape enabling common transcription factors to act on genes involved in ossification [9].…”