Growth Properties and Pluripotency Marker Expression of Spontaneously Formed Three-dimensional Aggregates of Human Adipose-derived Stem Cells

Abstract

Background and Objectives:Recent findings suggest that therapeutic potential of mesenchymal stem cells (MSCs) could be increased through aggregation into three-dimensional (3D) bodies, and different culture methods have been employed to obtain 3D spheroids of MSCs. In the current study we report accidentally encountered spontaneous formation of adipose-derived stem cell (ASC) bodies in standard ASC culture of a single donor.Methods and Results:Human ASCs from passages 1 to 3, cultured in a medium containing 5% autologous serum (AS), spontaneously clustered and formed floating 3D bodies. After a transfer of floating ASC bodies onto new adherent plastic dish, they attached to the surface and gradual migration of spindle-shaped ASCs out of the bodies was detected. A substitution of AS with allogeneic sera did not hinder this ability, but commercial medium containing fetal bovine serum delayed the process. Substantial part of ASCs surrounding transferred ASC bodies showed alkaline phosphatase (AP) activity, while ASC aggregates were AP negative. Similar 3D bodies formed when ASCs were grown on an uncoated glass surface. These ASC aggregates as well as clusters of ASCs, where formation of the 3D bodies is initiated, expressed pluripotency marker NANOG, but the expression of OCT4A was not detected.Conclusions:Obtained results suggest that spontaneously formed ASC aggregates may represent a more primitive cell subpopulation within the individual ASC culture. The ability to form 3D aggregates, the expression of NANOG, and the lack of the AP activity may be used to enrich ASC cultures with potentially more primitive cells serving as an excellent basis for therapeutic applications.

Cyclical aggregation extends in vitro expansion potential of human mesenchymal stem cells

“…Cyclical aggregation-planar expansion is necessary because MSCs lose function during planar expansion but are functionally enhanced during aggregation culture. Yet aggregated cells proliferate slower than planer cells [40][41][42] . Additionally, improvements of cellular properties have been shown to last one passage beyond aggregation 42 .…”

“…For example, Yang et al observed that passaging MSCs resulted in loss of cell shape and function as early as passage 5 33 . Previously, it has been shown that aggregation of ASCs results in the retention of stem cell morphology 41 . This study demonstrates that the cyclical aggregation likewise maintained the spindle morphology.…”

Cellular spheroids obtained from mesenchymal stem cells derived from bone marrow and limb muscle of early human embryo

“…No Oct 4 expression was revealed in 2D or 3D cultures of adipose derived human MSCs. Nanog expression was considerably increased in spheroids compared to 2D cultures (Bogdanova Jatniece et al, 2014). The authors have noticed that simultaneous expression of all three tran scription factors is required for pluripotency status.…”

“…The expres sion of undifferentiated ESC markers in MSCs proba bly explains their higher differentiation potential (Wu et al, 2013). Enhanced expression of these markers in spheroids compared to monolayer cultures suggests that they are primitive subpopulations with multipo tent potential (Bogdanova Jatniece et al, 2014;Guo et al, 2014).…”

“…It has been demonstrated that MSC spheroids exhibit increased anti inflammation effect, increased differentiation potential, and enhanced expression of cytokines and pluripotent genes. It facilitates the understanding of organogenesis mechanisms and their application in the regenerative medicine (Alimperti et al, 2014;Bogdanova Jatniece et al, 2014;Guo et al, 2014;Murphy et al, 2014;Xiao et al, 2014;Li et al, 2015).…”