Isolation and Characterization of Multipotent Mesenchymal Stem Cells Adhering to Adipocytes in Canine Bone Marrow

Abstract

The ceiling culture method has been used to isolate mature adipocytes from adipose tissue that can be dedifferentiated into fibroblastic cells, also known as dedifferentiated fat (DFAT) cells that self-renew and are multipotent, with much higher homogeneity and colony-forming efficiency than those of adipose tissue-derived mesenchymal stem cells. We cultured adipocytes from canine bone marrow using this technique, with the expectation of obtaining DFAT cells. However, contrary to our expectations, continuous monitoring of ceiling cultures by time-lapse microscopy revealed many small cells adhering to adipocytes that proliferated rapidly into cells with a fibroblastic morphology and without any dedifferentiation from adipocytes. We named these cells bone marrow peri-adipocyte cells (BM-PACs) and demonstrated the multipotent properties of BM-PACs compared to that of conventionally cultured canine bone marrow mesenchymal stem cells (BMMSCs). BM-PACs showed significantly greater clonogenicity and proliferation ability than BMMSCs. An in vitro trilineage differentiation assay revealed that BM-PACs possess adipogenic, osteogenic, and chondrogenic capacities superior to those of BMMSCs. Flow cytometric analysis revealed that the expression of CD73, which plays an important role in cell growth and differentiation, was significantly higher in BM-PACs than in BMMSCs. These results indicate that canine BM-PACs have stem cell characteristics that are superior to those of BMMSCs, and that these mesenchymal stem cells (MSCs) appear to be a feasible source for cell-based therapies in dogs.

Comparison of the effect of growth factors on chondrogenesis of canine mesenchymal stem cells

“…Canine BM-PACs were cultured according to a previously described method [22] with modifications in the composition of the growth medium after ceiling culture. Briefly, bone marrow aspirated from the proximal humerus was subjected to density gradient centrifugation with Ficoll-Paque (GE Healthcare, Little Chalfont, U.K.).…”

“…However, previous studies in canine MSCs have failed to demonstrate sufficient chondrogenic potential with abundant ECMs such as proteoglycans and type II collagen [4, 17, 20, 30, 31]. We previously established novel methods of culturing canine MSCs named bone marrow peri-adipocyte cells (BM-PACs), isolated from cells adhering to mature adipocytes in bone marrow [22]. BM-PACs showed superior proliferative capability and multipotency compared to those of conventional bone marrow-derived MSCs and can be a promising cell source for regenerative therapy in dogs.…”

Substantial Overview on Mesenchymal Stem Cell Biological and Physical Properties as an Opportunity in Translational Medicine

“…In this regard, Devireddy et al, [9] demonstrated that PDGF and bFGF synergistically promoted the growth and proliferation of adipose tissue-derived cMSC while TGF-ß1, that normally facilitates hMSC proliferation, decreased cMSC population (Table 1). cMSC showed higher CFU potential vs. hMSC, in addition to the enhanced proliferation capacity of synovium and adipose tissue cMSC [8,10] suggesting that these cells could represent a promising source for canine cartilage regeneration and for their utilization as a clinical model for human medicine.…”

Manufacturing and Banking Canine Adipose-Derived Mesenchymal Stem Cells for Veterinary Clinical Application

“…At present, in human clinical medicine, a variety of clinical trials with human MSCs are ongoing for the treatment of immunological diseases and degenerative diseases [6][7][8][9]. Currently, it has been reported that MSCs could be isolated from various tissues other than bone marrow [10], such as adipose [11], umbilical cord [12], dental pulp [13], placenta [14] and amniotic membrane [15]. Adipose-derived Mesenchymal stem cells (AD-MSCs) have gained in popularity due to the ease and abundance of harvesting adipose tissue, greater capacity of self-renewal multipotency and paracrine immunomodulatory [11,[16][17][18].…”