天井の培養における成熟脂肪細胞の脱分化を経てヒト脂肪幹細胞の生成

In vitro dedifferentiation of mature adipocytes is achieved through a technique called ceiling culture¹. Because of their natural tendency to float in aqueous solutions, isolated mature adipocytes adhere to the surface of an inverted flask fully filled with culture medium. Over a few days, cells modify their spherical morphology and become fibroblast-like cells. The resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent². Research articles on adipocyte dedifferentiation, especially on human cells, are limited. However, they have already provided interesting information regarding multipotency, cell phenotype and replicative capacity of DFAT cells². Mature adipocytes originating from various fat compartments have been successfully dedifferentiated including those originating from human visceral and subcutaneous adipose tissues^2-4. In addition to these depots, Kishimoto and collaborators sampled adipose tissue from the buccal fat pads and dedifferentiated adipocytes into DFAT cells⁵. Matsumoto and collaborators successfully generated subcutaneous DFAT cells from patients covering a wide range of ages, and the majority of cells had a high proliferative rate and less than 6% of senescence even after 10 passages in culture².

DFAT cells have been successfully re-differentiated into several lineages, including adipogenic, osteogenic, chondrogenic and neurogenic lineages^2,3,6. These cells express several embryonic stem cell markers such as Nanog and the four identified pluripotent factors Oct4, c-myc, Klf4 and Sox2³. They also express markers specific to each of the three germ layers⁷. In addition, DFAT cells are similar to Bone Marrow-derived Mesenchymal Stem Cells (BM-derived MSC) based on their epigenetic signature³. Exploiting the stem cell capacity of DFAT cells, many groups have investigated their potential to treat or improve various diseases^8,9. Improvements of pathologic conditions, such as infracted cardiac tissue, spinal cord injury and urethral sphincter dysfunction, have been observed with DFAT cell injections in rat models of disease^10-12.

In addition to the stem cell properties of DFAT cells, they may represent a new cellular model for adipocyte physiology studies. The 3T3-L1 cell line is often used for this purpose as these cells differentiate into adherent, lipid-storing adipocytes under adipogenic stimulation¹³. However, these cells originate from mouse embryo tissue¹³. Also, depot-specificity cannot be investigated with this model and it may not fully reflect human adipocyte physiology¹⁴. Other laboratories work with isolated adipose cells from murine fat depots, but fat distribution is not dimorphic in mice and anatomical configuration of the rodent’s abdominal cavity prevents from extrapolating directly to humans¹⁵. In order to study adipocytes in the context of the physiopathology of human obesity, consideration of body fat distribution and fat depot-specific differences has become essential¹⁶. Some limitations of primary preadipocyte cultures, including cell quantities obtained from adipose tissue biopsy samples and their senescence after a few passages in culture, created the need for alternate models. Perrini and collaborators investigated depot-specificity in gene expression of DFAT cells originating from visceral and subcutaneous fat and compared them to adipose-derived stem cells (ASC) from the same fat depot. They demonstrated that differences in gene expression and function where mainly found between depots than between cell types, suggesting that DFAT cells are physiologically close to ASC from the same depot. DFAT cells may represent an interesting alternative to available models for studies on fat distribution in the pathophysiology of human obesity. Moreover, ceiling culture is a promising method to obtain adult stem cells for tissue engineering purposes.

Here, we describe collagenase digestion, a widely-used technique to isolate mature adipocytes from the subcutaneous and/or visceral fat samples¹⁷, and the subsequent steps to perform ceiling culture and dedifferentiate these cells into multipotent, fibroblast-like cells.