Comparing the Osteogenic Potential and Bone Regeneration Capacities of Dedifferentiated Fat Cells and Adipose-Derived Stem Cells In Vitro and In Vivo: Application of DFAT Cells Isolated by a Mesh Method

Abstract

Background: We investigated and compared the osteogenic potential and bone regeneration capacities of dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs). Method: We isolated DFAT cells and ASCs from GFP mice. DFAT cells were established by a new culture method using a mesh culture instead of a ceiling culture. The isolated DFAT cells and ASCs were incubated in osteogenic medium, then alizarin red staining, alkaline phosphatase (ALP) assays, and RT-PCR (for RUNX2, osteopontin, DLX5, osterix, and osteocalcin) were performed to evaluate the osteoblastic differentiation ability of both cell types in vitro. In vivo, the DFAT cells and ASCs were incubated in osteogenic medium for four weeks and seeded on collagen composite scaffolds, then implanted subcutaneously into the backs of mice. We then performed hematoxylin and eosin staining and immunostaining for GFP and osteocalcin. Results: The alizarin red-stained areas in DFAT cells showed weak calcification ability at two weeks, but high calcification ability at three weeks, similar to ASCs. The ALP levels of ASCs increased earlier than in DFAT cells and showed a significant difference (p < 0.05) at 6 and 9 days. The ALP levels of DFATs were higher than those of ASCs after 12 days. The expression levels of osteoblast marker genes (osterix and osteocalcin) of DFAT cells and ASCs were higher after osteogenic differentiation culture. Conclusion: DFAT cells are easily isolated from a small amount of adipose tissue and are readily expanded with high purity; thus, DFAT cells are applicable to many tissue-engineering strategies and cell-based therapies.

The transdifferentiation of human dedifferentiated fat cells into fibroblasts: An in vitro experimental pilot study

“…[ 29 ] Adipose-derived stem cells (ASCs), which exhibit multilineage potential comparable to that of BMDCs, offer the advantage of easy accessibility to mature adipose tissue. [ 15 , 16 ] Nonetheless, ASCs are heterogeneous and stem from the nonadipocyte fraction within the adipose tissue, including the stromal-vascular fraction. Moreover, early passaged ASCs contain contaminating endothelial cells, smooth muscle cells, and pericytes.…”

“…High-purity cell sources are imperative for harnessing these stem cells for widespread clinical use. [ 15 , 16 , 20 , 30 ] Mature adipocytes constitute the majority of adipose tissue cells and are typically considered stable, having reached terminal differentiation. [ 20 ] However, Yagi et al [ 17 ] established a preadipocyte cell line, termed DFAT cells, derived from mature adipocytes of mice, demonstrating that these cells possess vigorous proliferative activity and have the potential to differentiate into various mesenchymal tissue lineages, such as BMDCs.…”

“…This unique property of DFAT cells stems from their isolation by ceiling culture, which exploits adipocyte buoyancy. [ 15 , 16 , 21 ] Consequently, DFAT cells are considered to be a homogeneous cell population, underpinning their potential utility in various clinical applications.…”

“…Similar to bone marrow-derived cells (BMDCs), DFAT cells have been demonstrated to possess the capacity for differentiating into diverse mesenchymal tissue lineages. [15,16,[18][19][20][21] Taking inspiration from this context, we conducted an in vitro study to explore the feasibility of using mature adipocytes to induce fibroblast-like characteristics in the adipose tissue and transdifferentiate them into skin tissue for skin regeneration in patients with skin defects.…”

“…[ 14 ] However, these stem cells are heterogeneous due to their mixed location within the stromal compartment, which exists in small quantities and limits their yield. [ 15 , 16 ] Recently, mature adipocytes, comprising approximately 40% of adipose tissue, have emerged as a potential source. After removing lipids from these cells, they adopt a fibroblast-like form and exhibit the ability to redifferentiate, offering relative homogeneity and high utility in research.…”