Supplementation of Strontium to a Chondrogenic Medium Promotes Chondrogenic Differentiation of Human Dedifferentiated Fat Cells

Abstract

Dedifferentiated fat cells (DFAT cells) isolated from adipose tissue have been demonstrated to differentiate into chondrogenic cells in vitro. Nevertheless, an efficient method to facilitate its chondrogenic differentiation is still unexplored, hampering the extensive application of these cells in cartilage regeneration therapies. Here we provide the evidence that supplementation of strontium ions (Sr) in a chondrogenic medium (CM) significantly promotes early chondrogenic differentiation of DFAT cells. Human DFAT cells and the mesenchymal stem cell line (RCB2153) were subjected to the CM supplemented with/without Sr. After 14 days, alcian blue staining intensity significantly increased in DFAT cells, but not in RCB2153, subjected to CM with Sr. mRNA expression analysis revealed that the CM with 1.5 mM Sr increased the expression of chondrogenic marker, collagen type 2 alpha 1, whereas there was no significant change in osteogenic markers, collagen type 1 alpha 1, runt-related transcription factor 2, and osteocalcin, and hypertrophic chondrogenic marker, collagen type 10 alpha 1. Inhibitors for extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and calcium-sensing receptor (CaSR) pathways significantly diminished the alcian blue staining intensity, providing the first evidence that these signal pathways are associated with chondrogenic differentiation of DFAT cells. CaSR and ERK1/2 pathways independently induced Sr-mediated early chondrogenic differentiation. These results suggest that Sr supplementation into the CM may provide a powerful platform for preparing chondrogenically differentiated DFAT cells for cartilage regeneration.

Dedifferentiated Fat (DFAT) cells: A cell source for oral and maxillofacial tissue engineering

“…After 7 days, fat cells have attached to the ceiling; the medium is now removed and the flask is re‐inverted (i.e., the ceiling now becomes the floor of the flask). Fresh medium is added to barely cover the bottom of the flask to allow cells to continue their expansion (Kaida, Honda, Hashimoto, Tanaka, & Baba, ; Kishimoto et al., ; Matsumoto et al., ; Okita et al., ; Sakamoto, Hashimoto, Kishimoto, Honda, & Matsumoto, ; Sakuma et al., ). Adipocytes that have attached to the inner surface of the flask during the ceiling culture divide asymmetrically and produce fibroblast‐like cells.…”

“…The cell transplants were harvested after 3 months and were stained by safranin O and alcian blue to evaluate the chondrogenic differentiation of DFAT cells. A reticulated structure, rather than dense and large cartilage‐like tissue, was observed (Okita et al., ). Other groups have reported that fat pads with blood vessels were formed when DFAT cells were injected subcutaneously in the sternum of mice (Nobusue et al., ).…”

“…Jumabay and colleagues isolated human DFAT cells without using the ceiling culture method. Human DFAT cells differentiated into adipocytes (Matsumoto et al, 2008;Poloni et al, 2012;Sakuma et al, 2009;Watson et al, 2014), chondrocytes (Azumi, Honda, Kishimoto, Hashimoto, & Matsumoto, 2016;Jumabay et al, 2014;Matsumoto et al, 2008;Okita et al, 2015;Poloni et al, 2012;Sakuma et al, 2009), osteoblasts (Hao et al, 2017;Jumabay et al, 2014;Kaida et al, 2015;Matsumoto et al, 2008;Poloni et al, 2012;Sakamoto et al, 2015;Watson et al, 2014), smooth muscle-like cells (Sakuma et al, 2009), and neurogeniclineage cells (Poloni et al, 2012) under appropriate culture conditions in vitro.…”

“…When DFAT cells were transplanted to a bladder injury model in mice, Our group formed cell sheets of human DFAT cells cultured in chondrogenic induction medium (CIM) and subcutaneously injected these cells into SCID mice (Okita et al, 2015). The cell transplants were harvested after 3 months and were stained by safranin O and alcian blue to evaluate the chondrogenic differentiation of DFAT cells.…”

“…This positive effect of strontium was not observed in human BMSCs. We consider that it is useful to add 1.5 mM of strontium to the medium when B-DFAT cells are cultured for cartilage tissue engineering (Okita et al, 2015). Kou et al cultured B-DFAT cells in AIM.…”