11 research outputs found
Genome-wide analysis of gene expression during adipogenesis in human adipose-derived stromal cells reveals novel patterns of gene expression during adipocyte differentiation
We have undertaken an in-depth transcriptome analysis of adipogenesis in human adipose-derived stromal cells
(ASCs) induced to differentiate into adipocytes in vitro. Gene expression was assessed on days 1, 7, 14 and 21
post-induction and genes differentially expressed numbered 128, 218, 253 and 240 respectively. Up-regulated
genes were associated with blood vessel development, leukocyte migration, as well as tumor growth, invasion
and metastasis. They also shared common pathways with certain obesity-related pathophysiological conditions.
Down-regulated genes were enriched for immune response processes. KLF15, LMO3, FOXO1 and ZBTB16 transcription
factors were up-regulated throughout the differentiation process. CEBPA, PPARG, ZNF117, MLXIPL,
MMP3 and RORB were up-regulated only on days 14 and 21, which coincide with the maturation of adipocytes
and could possibly serve as candidates for controlling fat accumulation and the size of mature adipocytes. In summary,
we have identified genes that were up-regulated only on days 1 and 7 or days 14 and 21 that could serve as
potential early and late-stage differentiation markers.The South African Medical Research
Council in terms of the SAMRC's Flagship Award Project SAMRC-RFAUFSP-
01-2013/STEM CELLS, the SAMRC Extramural Stem Cell Research
and Therapy Unit, the National Research Foundation of South Africa
(grant no. 86942), the National Health Laboratory Services Research
Trust (grant no. 94453) and the Institute for Cellular and Molecular
Medicine of the University of Pretoria.http://www.elsevier.com/locate/scram2016African Language
Novel flow cytometric approach for the detection of adipocyte subpopulations during adipogenesis
The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of
human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack
of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool
of MSCs. Cluster of differentiation (CD)36 plays an important role in the formation of intracellular lipid
droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to
develop a reproducible quantitative method to study adipocyte differentiation by comparing two lipophilic
dyes [Nile Red (NR) and Bodipy 493/503] in combination with CD36 surface marker staining. We identified
a subpopulation of adiposederived
stromal cells that express CD36 at intermediate/high levels and show that
combining CD36 cell surface staining with neutral lipidspecific
staining allows us to monitor differentiation
of adiposederived
stromal cells that express CD36 during adipocyte differentiation in vitro.
The gradual increase of CD36 NR cells during the 21 day adipogenesis induction
period correlated with upregulation of adipogenesisassociated
gene expression.http://www.jlr.org2017-04-30hb2016Immunolog
Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion
Reference gene expression in adipose-derived stromal cells undergoing adipogenic differentiation
Adipose-derived stromal cells (ASCs) are becoming increasingly attractive as cellular therapy products. Their
differentiation potential, the secretion of growth and differentiation factors, and the ability to cryopreserve the cells
over extended periods are important features. Changes in experimental conditions result in changes in gene expression, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an important tool
for measuring these changes. There is, however, the potential to introduce technical bias in the process, which can be
diminished through the selection of stable reference genes (RGs). Using geNorm software, in this in vitro study we
explore the effects that adipogenic differentiation for a 21 day induction period, cryopreservation (freshly isolated
ASCs or previously cryopreserved/frozen ASCs), and culture medium supplementation (fetal bovine serum vs.
pooled human platelet lysate) have on the stability of 11 RGs. We found that RG stability is markedly affected by the
different experimental conditions. Of the RGs assessed, YWHAZ, HPRT, TBP, andACTB were stably expressed genes
under all experimental conditions. We recommend that a panel of stable RGs should be selected before studying gene
expression during adipogenesis, and that this is based on the experimental condition(s) being investigated.https://www.liebertpub.com/tecpm2020Immunolog
Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion
Pooled human platelet lysate (pHPL) has been used to expand adipose-derived stromal cells
(ASCs) and can be formulated using fresh or expired buffy coats (BCs) which are then
resuspended in either plasma or an additive solution. Not much is known about the effects
that expired products and additive solutions have on ASC expansion, and the need for quality
control and release criteria has been expressed. This pilot study compared proliferation, cell
size, morphology and immunophenotype of ASCs expanded in the different pHPL alternatives
versus foetal bovine serum (FBS). Quality control criteria were assessed prior to and during the
manufacture of the pHPL alternatives. ASCs were then expanded in 1%, 2.5%, 5% or 10% of the
different pHPL alternatives or in 10% FBS. Cell size, morphology, cell number and immunophenotype
were measured using microscopy and flow cytometry. The majority of the pHPL
alternatives were within the recommended ranges for the quality control criteria. ASCs
expanded in the pHPL alternatives were smaller in size, displayed a tighter spindle-shaped
morphology, increased cell growth and had a similar immunophenotype (with the exception of
CD34 and CD36) when compared to ASCs expanded in FBS. Here we report on the effects that
expired BC products and additive solutions have on ASC expansion. When taken together, our
findings indicate that all of the pHPL alternatives can be considered to be suitable replacements
for FBS for ASC expansion, and that expired BC products can be used as an alternative to
fresh BC products.Grants from the South African
Medical Research Council Flagship Awards Project SAMRC-RFA-UFSP-
01-2013/STEM CELLS, the SAMRC Extramural Unit for Stem Cell
Research and Therapy and the Institute for Cellular and Molecular
Medicine of the University of Pretoria. The National Research
Foundation of South Africa also provided funding.http://www.tandfonline.com/loi/iplt20am2018Immunolog