Mei Kuen Tang

CD146+ Human Umbilical Cord Perivascular Cells Maintain Stemness under Hypoxia and as a Cell Source for Skeletal Regeneration

The human umbilical cord perivascular cells (HUCPVCs) have been considered as an alternative source of mesenchymal progenitors for cell based regenerative medicine. However, the biological properties of these cells remain to be well characterized. In the present study, HUCPVCs were isolated and sorted by CD146+ pericyte marker. The purified CD146+ HUCPVCs were induced to differentiate efficiently into osteoblast, chondrocyte and adipocyte lineages in vitro. Six weeks following subcutaneous transplantation of CD146+ HUCPVCs-Gelfoam-alginate 3D complexes in severe combined immunodeficiency (SCID) mice, newly formed bone matrix with embedded osteocytes of donor origin was observed. The functional engraftment of CD146+ HUCPVCs in the new bone regenerates was further confirmed in a critical-sized bone defect model in SCID mice. Hypoxic conditions suppressed osteogenic differentiation while increased cell proliferation and colony-forming efficiency of CD146+ HUCPVCs as compared to that under normoxic conditions. Re-oxygenation restored the multi-differentiation potential of the CD146+ HUCPVCs. Western blot analysis revealed an upregulation of HIF-1α, HIF-2α, and OCT-4 protein expression in CD146+ HUCPVCs under hypoxia, while there was no remarkable change in SOX2 and NANOG expression. The gene expression profiles of stem cell transcription factors between cells treated by normoxia and hypoxic conditions were compared by PCR array analysis. Intriguingly, PPAR-γ was dramatically downregulated (20-fold) in mRNA expression under hypoxia, and was revealed to possess a putative binding site in the Hif-2α gene promoter region. Chromatin immunoprecipitation assays confirmed the binding of PPAR-γ protein to the Hif-2α promoter and the binding was suppressed by hypoxia treatment. Luciferase reporter assay showed that the Hif-2α promoter activity was suppressed by PPAR expression. Thus, PPAR-γ may involve in the regulation of HIF-2α for stemness maintenance and promoting the expansion of CD146+ HUCPVCs in response to hypoxia. CD146+ HUCPVCs may serve as a potential autologous cell source for bone regeneration.

Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death

In this study, we used comparative proteomics to identify proteins that were involved in the regulation of interdigital cell death. The protein profiles of embryonic day (E) 12.5 and 13.5 mouse hindlimb interdigital tissues were compared to identify proteins that were differentially expressed. The interdigital cells are irreversibly committed to programmed cell death (PCD) at E13.5, whereas they are developmentally plastic at E12.5. We established that protein disulfide isomerase (PDI) expression was up‐regulated at E13.5, while peroxiredoxin 1 (Prdx1) expression was down‐regulated at this time point. Semiquantitative reverse transcriptase‐polymerase chain reaction and Western blot analyses confirmed the data obtained from the two‐dimensional electrophoresis gels. Furthermore, we were able to up‐regulate PDI expression by manipulating the E12.5 interdigital tissues to die during culture, although this up‐regulation was not possible when cell survival was promoted. In addition, we could inhibit interdigital cell death and expression of proapoptotic genes (Bmp‐4 and Bambi) by treating interdigital tissues with PDI antibodies and bacitracin (a PDI enzyme inhibitor). These findings suggested that PDI was involved in the activation and maintenance of interdigital cell death. Conversely, we determined that Prdx1 expression was maintained when interdigital cultures were manipulated to survive but down‐regulated when the cultures were permitted to die. The result suggested that Prdx1 was involved in maintaining interdigital cell survival. However, we were unable to induce interdigital cell death by means of RNA interference‐mediated silencing of Prdx1 expression, indicating that Prdx1 down‐regulation is not sufficient for PCD to occur. Proteomic analysis of the Prdx1 knock‐down cells revealed that the level of NF‐kappaB inhibitor epsilon (IκBε) was dramatically reduced. Furthermore, we found an increase in NFκB activation and reactive oxygen species (ROS) levels in the cytoplasm as a result of Prdx1 knockdown. We also found that silencing Prdx1 made the interdigital cells more susceptible to ROS‐induced cell death. Taken together, our study identifies two new players in interdigital cell death and highlights that PCD is regulated by a delicate balance of proapoptotic and survival‐promoting activities. Developmental Dynamics 233:266–281, 2005.

Cardiogenol C can induce Mouse Hair Bulge Progenitor Cells to Transdifferentiate into Cardiomyocyte-like Cells

BackgroundHair bulge progenitor cells (HBPCs) are multipotent stem cells derived from the bulge region of mice vibrissal hairs. The purified HBPCs express CD34, K15 and K14 surface markers. It has been reported that HBPCs could be readily induced to transdifferentiate into adipocytes and osteocytes. However, the ability of HBPCs to transdifferentiate into cardiomyocytes has not yet been investigated.Methodology/Principal FindingsThe cardiomyogenic potential of HBPCs was investigated using a small cell-permeable molecule called Cardiogenol C. We established that Cardiogenol C could induce HBPCs to express transcription factors GATA4, Nkx2.5 and Tbx5, which are early specific markers for pre-cardiomyogenic cells. In prolonged cultures, the Cardiogenol C-treated HBPCs can also express muscle proteins, cardiac-specific troponin I and sarcomeric myosin heavy chain. However, we did not observe the ability of these cells to functionally contract. Hence, we called these cells cardiomyocyte-like cells rather than cardiomyocytes. We tried to remedy this deficiency by pre-treating HBPCs with Valproic acid first before exposing them to Cardiogenol C. This pretreatment inhibited, rather than improved, the effectiveness of Cardiogenol C in reprogramming the HBPCs. We used comparative proteomics to determine how Cardiogenol C worked by identifying proteins that were differentially expressed. We identified proteins that were involved in promoting cell differentiation, cardiomyocyte development and for the normal function of striated muscles. From those differentially expressed proteins, we further propose that Cardiogenol C might exert its effect by activating the Wnt signaling pathway through the suppression of Kremen1. In addition, by up-regulating the expression of chromatin remodeling proteins, SIK1 and Smarce1 would initiate cardiac differentiation.Conclusions/SignificanceIn conclusion, our CD34+/K15+ HBPCs could be induced to transdifferentiate into cardiomyocyte-like cells using a small molecule called Cardiogenol C. The process involves activation of the Wnt signaling pathway and altered expression of several key chromatin remodeling proteins. The finding is clinically significant as HBPCs offer a readily accessible and autologous source of progenitor cells for cell-based therapy of heart disease, which is one of major killers in developed countries.

Promyelocytic Leukemia (PML) Protein Plays Important Roles in Regulating Cell Adhesion, Morphology, Proliferation and Migration

PML protein plays important roles in regulating cellular homeostasis. It forms PML nuclear bodies (PML-NBs) that act like nuclear relay stations and participate in many cellular functions. In this study, we have examined the proteome of mouse embryonic fibroblasts (MEFs) derived from normal (PML+/+) and PML knockout (PML−/−) mice. The aim was to identify proteins that were differentially expressed when MEFs were incapable of producing PML. Using comparative proteomics, total protein were extracted from PML−/− and PML+/+ MEFs, resolved by two dimensional electrophoresis (2-DE) gels and the differentially expressed proteins identified by LC-ESI-MS/MS. Nine proteins (PML, NDRG1, CACYBP, CFL1, RSU1, TRIO, CTRO, ANXA4 and UBE2M) were determined to be down-regulated in PML−/− MEFs. In contrast, ten proteins (CIAPIN1, FAM50A, SUMO2 HSPB1 NSFL1C, PCBP2, YWHAG, STMN1, TPD52L2 and PDAP1) were found up-regulated. Many of these differentially expressed proteins play crucial roles in cell adhesion, migration, morphology and cytokinesis. The protein profiles explain why PML−/− and PML+/+ MEFs were morphologically different. In addition, we demonstrated PML−/− MEFs were less adhesive, proliferated more extensively and migrated significantly slower than PML+/+ MEFs. NDRG1, a protein that was down-regulated in PML−/− MEFs, was selected for further investigation. We determined that silencing NDRG1expression in PML+/+ MEFs increased cell proliferation and inhibited PML expression. Since NDRG expression was suppressed in PML−/− MEFs, this may explain why these cells proliferate more extensively than PML+/+ MEFs. Furthermore, silencing NDRG1expression also impaired TGF-β1 signaling by inhibiting SMAD3 phosphorylation.

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-β/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.

Integrative Analysis of the Developing Postnatal Mouse Heart Transcriptome

In mammals, cardiomyocytes rapidly proliferate in the fetus and continue to do so for a few more days after birth. These cardiomyocytes then enter into growth arrest but the detailed molecular mechanisms involved have not been fully elucidated. We have addressed this issue by comparing the transcriptomes of 2-day-old (containing dividing cardiomyocytes) with 13-day-old (containing growth arrested cardiomyocytes) postnatal mouse hearts. We performed comparative microarray analysis on the heart tissues and then conducted Functional annotation, Gene ontology, KEGG pathway and Gene Set enrichment analyses on the differentially expressed genes. The bioinformatics analysis revealed that gene ontology categories associated with the “cell cycle”, “DNA replication”, “chromosome segregation” and “microtubule cytoskeleton” were down-regulated. Inversely, “immune response”, “extracellular matrix”, “cell differentiation” and “cell membrane” were up-regulated. Ingenuity Pathways Analysis (IPA) has revealed that GATA4, MYH7 and IGF1R were the key drivers of the gene interaction networks. In addition, Regulator Effects network analysis suggested that TASP1, TOB1, C1orf61, AIF1, ROCK1, TFF2 and miR503-5p may be acting on the cardiomyocytes in 13-day-old mouse hearts to inhibit cardiomyocyte proliferation and G1/S phase transition. RT-qPCR was used to validate genes which were differentially expressed and genes that play a prominent role in the pathways and interaction networks that we identified. In sum, our integrative analysis has provided more insights into the transcriptional regulation of cardiomyocyte exit from the cell cycle during postnatal heart development. The results also pinpoint potential regulators that could be used to induce growth arrested cardiomyocytes to proliferate in the infarcted heart.

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).

BRE plays an essential role in preventing replicative and DNA damage-induced premature senescence

The BRE gene, alias BRCC45, produces a 44 kDa protein that is normally distributed in both cytoplasm and nucleus. In this study, we used adult fibroblasts isolated from wild-type (WT) and BRE knockout (BRE−/−) mice to investigate the functional role of BRE in DNA repair and cellular senescence. We compared WT with BRE−/− fibroblasts at different cell passages and observed that the mutant fibroblasts entered replicative senescence earlier than the WT fibroblasts. With the use of gamma irradiation to induce DNA damage in fibroblasts, the percentage of SA-β-Gal+ cells was significantly higher in BRE−/− fibroblasts compared with WT cells, suggesting that BRE is also associated with DNA damage-induced premature senescence. We also demonstrated that the gamma irradiation induced γ-H2AX foci, a DNA damage marker, persisted significantly longer in BRE−/− fibroblasts than in WT fibroblasts, confirming that the DNA repair process is impaired in the absence of BRE. In addition, the BRCA1-A complex recruitment and homologous recombination (HR)-dependent DNA repair process upon DNA damage were impaired in BRE−/− fibroblasts. Taken together, our results demonstrate a role for BRE in both replicative senescence and DNA damage-induced premature senescence. This can be attributed to BRE being required for BRCA1-A complex-driven HR DNA repair.