Neural crest-derived mesenchymal progenitor cells enhance cranial allograft integration

Abstract

Replacement of lost cranial bone (partly mesodermal and partly neural crest‐derived) is challenging and includes the use of nonviable allografts. To revitalize allografts, bone marrow‐derived mesenchymal stromal cells (mesoderm‐derived BM‐MSCs) have been used with limited success. We hypothesize that coating of allografts with induced neural crest cell‐mesenchymal progenitor cells (iNCC‐MPCs) improves implant‐to‐bone integration in mouse cranial defects. Human induced pluripotent stem cells were reprogramed from dermal fibroblasts, differentiated to iNCCs and then to iNCC‐MPCs. BM‐MSCs were used as reference. Cells were labeled with luciferase (Luc2) and characterized for MSC consensus markers expression, differentiation, and risk of cellular transformation. A calvarial defect was created in non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and allografts were implanted, with or without cell coating. Bioluminescence imaging (BLI), microcomputed tomography (μCT), histology, immunofluorescence, and biomechanical tests were performed. Characterization of iNCC‐MPC‐Luc2 vs BM‐MSC‐Luc2 showed no difference in MSC markers expression and differentiation in vitro. In vivo, BLI indicated survival of both cell types for at least 8 weeks. At week 8, μCT analysis showed enhanced structural parameters in the iNCC‐MPC‐Luc2 group and increased bone volume in the BM‐MSC‐Luc2 group compared to controls. Histology demonstrated improved integration of iNCC‐MPC‐Luc2 allografts compared to BM‐MSC‐Luc2 group and controls. Human osteocalcin and collagen type 1 were detected at the allograft‐host interphase in cell‐seeded groups. The iNCC‐MPC‐Luc2 group also demonstrated improved biomechanical properties compared to BM‐MSC‐Luc2 implants and cell‐free controls. Our results show an improved integration of iNCC‐MPC‐Luc2‐coated allografts compared to BM‐MSC‐Luc2 and controls, suggesting the use of iNCC‐MPCs as potential cell source for cranial bone repair.

An Evolutionary Conserved Signaling Network Between Mouse and Human Underlies the Differential Osteoskeletal Potential of Frontal and Parietal Calvarial Bones

“…Furthermore, our findings also align with two previous studies showing that either an ecto-mesenchymal stem cell (eMSC) population-derived from human embryonic stem cells through a neural crest intermediate or induced human neural crest cell-mesenchymal progenitor cells (iNCC-MPCs) promoted new and enhanced bone formation in murine calvarial defects as compared to mesoderm-derived bone marrow mesenchymal stem cells (BM-MSCs) (Glaeser et al, 2021;Srinivasan et al, 2021). Taken all together, these findings strongly suggest the importance of taking in account the cell-origin when selecting a cell-source for bone regeneration applications.…”

Directing iPSC Differentiation into iTenocytes using Combined Scleraxis Overexpression and Cyclic Loading

“…The Livak method was used to calculate ΔΔCt values and fold change was calculated as 2 −ΔΔCt , as previously described and published. 30; 31; 43–46…”

“…Human bone marrow was acquired from Lonza (Allendale, NJ) and BM-MSCs were isolated as previously reported. [30][31][32] Briefly, bone marrow was overlayed on Ficoll-paque density gradient medium (GE healthcare, Chicago, IL), washed twice with phosphate buffered saline (PBS), plated at a density of 2x10 5 /cm 2 (Corning, Corning, NY), and incubated overnight at 37 o C/5% CO 2 . After 48h, non-adherent cells were discarded, washed with phosphate buffered saline (PBS), and culture medium was added, composed of 2mM L-glutamine, 1% antibiotic antimycotic solution (AAS) in Dulbecco's modified eagle medium (DMEM, all from Thermofisher, Waltham, MA), and 20% fetal bovine serum (FBS, GeminiBio, West Sacramento, CA).…”

A Narrative Review of Cell-Based Approaches for Cranial Bone Regeneration

“…Although neural crest cells are very scarce in humans, they can be derived in large quantities from iPSCs [95]. Recently, iPSCs reprogrammed from human dermal fibroblasts were differentiated into neural crest cells and then into neural crest-derived mesenchymal progenitor cells [96]. These cells were seeded in a decellularized cranial allograft and implanted in defects created in the calvaria of non-obese diabetic/severe combined immunodeficient mice.…”