Directing stem cell fate by controlled RNA interference

Directing stem cell fate remains a major area of interest and also a hurdle to many, particularly in the field of regenerative medicine. Unfortunately, conventional methods of over-expressing inductive factors through the use of biochemical induction cocktails have led to sub-optimal outcomes. A potential alternative may be to adopt the opposite by selectively silencing genes or pathways that are pivotal to stem cell differentiation. Indeed, over recent years, there have been an increasing number of studies on directing stem cell fate through gene knockdown via RNA interference (RNAi). While the effectiveness of RNAi in controlling stem cell differentiation is evident from the myriad of studies, a chaotically vast collection of gene silencing targets have also been identified. Meanwhile, variations in methods of transfecting stem cells have also affected silencing efficiencies and the subsequent extent of stem cell differentiation. This review serves to unite the pioneers who have ventured into the emerging field of RNAi-enhanced stem cell differentiation by summarizing and evaluating the current approaches adopted in utilizing gene silencing to direct stem cell fate and their corresponding outcomes

Silencing BRE Expression in Human Umbilical Cord Perivascular (HUCPV) Progenitor Cells Accelerates Osteogenic and Chondrogenic Differentiation

<div><p>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 <i>BRE</i> expression, using <i>BRE</i>-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 <i>BRE</i>-silenced cells. <i>BRE</i>-silencing negatively regulated <i>OCT4</i>, <i>FGF5</i> and <i>FOXO1A</i>. <i>BRE</i>-silencing also altered the expression of epigenetic genes and components of the TGF-β/BMP and FGF signaling pathways which are crucially involved in maintaining stem cell self-renewal. Comparative proteomic profiling also revealed that <i>BRE</i>-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.</p></div

Thyroid hormone (T3) stimulates brown adipose tissue activation via mitochondrial biogenesis and MTOR-mediated mitophagy

10.1080/15548627.2018.1511263Autophagy115131-15

HUCPV cells differentiated into osteoblasts and chondrocytes, 3 and 4 weeks after induction, respectively.

<p>(A) Alizarin red S staining and immunofluorescence staining with pro-collagen type-I antibody were used to demonstrate osteogenic differentiation by HUCPV cells maintained in osteogenic (Ost) medium and control (Ctl) media for 3 weeks. (B) Alcian blue staining and immunofluorescence staining with SOX9 antibody were used to demonstrate chondrogenic differentiation in HUCPV cells cultured in chondrogenic (Chon) and control (Ctl) media for 4 weeks. The nuclei were counterstained with DAPI. N = 3 independent experiments.</p

Extraction and purification of HUCPV cells.

<p>(A) Representative picture of a human umbilical cord showing the umbilical vein (represented by solid circle) and umbilical arteries (represented by dashed circle). (B) Prior to treatment with collagenase, the umbilical blood vessel was ligated at both ends. (C) The primary HUCPV cells were isolated by collagenase digestion of the perivascular region of the ligated blood vessel.</p

Comparative proteomics analysis of differentially expressed proteins in BRE silenced HUCPV cells.

<p>A representative silver stained 2-DE gel of total protein extracted from HUCPV cells that had been transfected with <i>BRE</i>-siRNA. When compared with control 2-DE gels, the ESI-MS/MS analysis identified proteins that were up- and down-regulated as a result of silencing <i>BRE</i>. Cytoskeletal binding proteins were especially affected. N = 3 independent experiments.</p

BRE expressions in <i>BRE</i>-silenced HUCPV cells.

<p>(A) RT-qPCR and (B) immunofluorescence microscopy showing <i>BRE</i>-siRNAs could silence BRE expression in HUCPV cells. Our control <i>Ctl-siRNAs</i> did not affect BRE expression. For RT-qPCR, <i>BRE</i> expression was normalized to housekeeping gene <i>GAPDH</i>. The statistical difference of P values were determined by t-test and **p<0.01 were considered significantly different. The nuclei were counterstained with DAPI. N = 3 independent experiments.</p

BRE expressions during HUCPV cell differentiation.

<p>(A) RT-qPCR revealed that as HUCPV cells were induced to differentiate in osteogenic (Ost) and chondrogenic (chon) medium, <i>BRE</i> expression was down-regulated. <i>BRE</i> expression was normalized against <i>GAPDH</i>. The statistical difference of P values were determined by t-test; *p<0.05, **p<0.01 and *p<.05 were considered significantly different. (B) Immunofluorescence microscopy confirmed that BRE expression (red) was suppressed as the HUCPV cells were induced to form osteoblasts and chondroblasts. Control (Ctl) medium. Osteogenic (Ost) and chondrogenic (Chon) inducing medium. The nuclei were counterstained with DAPI. N = 3 independent experiments.</p

BRE and OCT4 expression in ESCs.

<p>(A–C) In the presence of LIF, BRE and OCT4 were strongly co-expressed in undifferentiated ESCs (yellow dotted outlines). (D) LIF was withdrawn from the culture for 24 hours to allow the ESCs to differentiate. This resulted in a reduction of BRE and OCT4 expression in the ESCs (white dotted outlines). N = 3 independent experiments.</p

Propose model of BRE function in HUPVC cells.

<p>BRE is normally involved in inhibiting HUPVC cell differentiation and the maintenance of OCT4 expression. When BRE expression is silenced, OCT4 expression is partially inhibited and HUPVC cell differentiation is accelerated following osteogenic or chondrogenic induction.</p