Growing Human Dermal Fibroblasts as Spheroids Renders Them Susceptible for Early Expression of Pluripotency Genes.

Suspension spheroid cultures of anchorage-dependent cell types have been widely used in cancer and stem cell research, as well as for producing organoids. It is believed that the 3-dimensional spheroid presents cells with a more physiological microenvironment to grow so that they behave more like cells in vivo, which is lacking in conventional 2-dimensional monolayer cultures. Recently, it has been reported that cancer cells grown as spheroids could express stem cell-associated genes. Hence, it is investigated whether normal mouse and human fibroblasts cultured as spheroids could also be induced to express stem cell-associated genes. The transcriptomes of human fibroblasts cultured as a monolayer and spheroids are compared and analyzed using real-time RT-qPCR, RNA-sequencing, and bioinformatics. The results reveal that the spheroid transcriptome resemble somatic cells being reprogramed into stem cells, including the induced expression of stemness-associated genes, increased expression of mesenchymal-to-epithelial transition-associated genes, and decreased expression of epithelial-to-mesenchymal transition-associated genes. In this context, it is hypothesized that during the process of spheroid formation, matrix-cell signaling is lost in favor of cell-cell contact signaling and that this subsequently increases the activity of the PI3K/Akt pathway that then upregulates Tbdx3 and stemness-associated genes.

Transient acid treatment cannot induce neonatal somatic cells to become pluripotent stem cells.

Currently, there are genetic- and chemical-based methods for producing pluripotent stem cells from somatic cells, but all of them are extremely inefficient.  However, a simple and efficient technique has recently been reported by Obokata et al (2014a, b) that creates pluripotent stem cells through acid-based treatment of somatic cells.  These cells were named stimulus-triggered acquisition of pluripotency (STAP) stem cells. This would be a major game changer in regenerative medicine if the results could be independently replicated. Hence, we isolated CD45 (+) splenocytes from five-day-old Oct4-GFP mice and treated the cells with acidified (pH 5.7) Hank's Balanced Salt Solution (HBSS) for 25 min, using the methods described by Obokata et al 2014c. However, we found that this method did not induce the splenocytes to express the stem cell marker Oct4-GFP when observed under a confocal microscope three to six days after acid treatment. qPCR analysis also confirmed that acid treatment did not induce the splenocytes to express the stemness markers Oct4, Sox2 and Nanog.  In addition, we obtained similar results from acid-treated Oct4-GFP lung fibroblasts. In summary, we have not been able to produce STAP stem cells from neonatal splenocytes or lung fibroblasts using the acid-based treatment reported by Obokata et al (2014a, b, c).

Silencing BRE expression in human umbilical cord perivascular (HUCPV) progenitor cells accelerates osteogenic and chondrogenic differentiation.

BRE is a multifunctional adapter protein involved in DNA repair, cell survival and stress response. To date, most studies of this protein have been focused in the tumor model. The role of BRE in stem cell biology has never been investigated. Therefore, we have used HUCPV progenitor cells to elucidate the function of BRE. HUCPV cells are multipotent fetal progenitor cells which possess the ability to differentiate into a multitude of mesenchymal cell lineages when chemically induced and can be more easily amplified in culture. In this study, we have established that BRE expression was normally expressed in HUCPV cells but become down-regulated when the cells were induced to differentiate. In addition, silencing BRE expression, using BRE-siRNAs, in HUCPV cells could accelerate induced chondrogenic and osteogenic differentiation. Hence, we postulated that BRE played an important role in maintaining the stemness of HUCPV cells. We used microarray analysis to examine the transcriptome of BRE-silenced cells. BRE-silencing negatively regulated OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and components of the TGF-ß/BMP and FGF signaling pathways which are crucially involved in maintaining stem cell self-renewal. Comparative proteomic profiling also revealed that BRE-silencing resulted in decreased expressions of actin-binding proteins. In sum, we propose that BRE acts like an adaptor protein that promotes stemness and at the same time inhibits the differentiation of HUCPV cells.