Generation of Two Multipotent Mesenchymal Progenitor Cell Lines Capable of Osteogenic, Mature Osteocyte, Adipogenic, and Chondrogenic Differentiation

Abstract

Mesenchymal progenitors differentiate into several tissues including bone, cartilage, and adipose. Targeting these cells in vivo is challenging, making mesenchymal progenitor cell lines valuable tools to study tissue development. Mesenchymal stem cells (MSCs) can be isolated from humans and animals; however, obtaining homogenous, responsive cells in a reproducible fashion is challenging. As such, we developed two mesenchymal progenitor cell (MPC) lines, MPC1 and MPC2, generated from bone marrow of male C57BL/6 mice. These cells were immortalized using the temperature sensitive large T-antigen, allowing for thermal control of proliferation and differentiation. Both MPC1 and MPC2 cells are capable of osteogenic, adipogenic, and chondrogenic differentiation. Under osteogenic conditions, both lines formed mineralized nodules, and stained for alizarin red and alkaline phosphatase, while expressing osteogenic genes including Sost, Fgf23, and Dmp1. Sost and Dmp1 mRNA levels were drastically reduced with parathyroid hormone, thus recapitulating in vivo responses. MPC cells secreted intact (iFGF23) and C-terminal (cFGF23) forms of the endocrine hormone FGF23, which was upregulated by 1,25 dihydroxy vitamin D (1,25D). Both lines also rapidly entered the adipogenic lineage, expressing adipose markers after 4 days in adipogenic media. MPC cells were also capable of chondrogenic differentiation, displaying increased expression of cartilage genes including aggrecan, Sox9, and Comp. With the ability to differentiate into multiple mesenchymal lineages and mimic in vivo responses of key regulatory genes/proteins, MPC cells are a valuable model to study factors that regulate mesenchymal lineage allocation as well as the mechanisms that dictate transcription, protein modification, and secretion of these factors.

The HIF-PHI BAY 85-3934 (Molidustat) Improves Anemia and Is Associated With Reduced Levels of Circulating FGF23 in a CKD Mouse Model

“…To build upon the concept of improved iron utilization reducing circulating FGF23 in vivo as we observed in the mice with CKD, we used an in vitro model of iron deficiency/hypoxia. The MPC2 conditional mesenchymal stem cell line ( 29 ) does not express osteocytic markers or mineralize until differentiated via culture in osteogenic media (see Materials and Methods and Supplemental Fig. S3), similar to primary mesenchymal stem cells, ( 40 ) whereas 3 weeks after differentiation, the cells robustly express the osteoblast/osteocyte transcripts encoding Fgf23, dentin matrix protein‐1 (Dmp1), type I collagen (Col1a1), and osteocalcin (Bglap) (Supplemental Fig.…”

“…The MSC cell line murine progenitor cells clone 2 (MPC2) ( 29 ) were cultured in αMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Thermo Fisher Scientific), 25 mM L‐glutamine, and 25 mM penicillin–streptomycin (Sigma‐Aldrich, St. Louis, MO, USA) at 33°C and 5% CO 2 to proliferate. Cells were plated at a density of 1.0 × 10 5 cells per mL in 6‐ or 12‐well plates and incubated overnight before being transferred to a 37°C incubator for osteogenic differentiation, with cultures in maintenance media supplemented with mineralization conditions of 4 mM beta‐glycerophosphate and 50 μg/mL ascorbic acid.…”