Oct-4/Sox-2 协同调控下游基因表达的分子机制

Oct-4属POU家族蛋白,是一类在动物早期胚胎发育过程中起重要作用的转录因子,参与维持细胞的全能性及未分化状态。Oct-4蛋白的主要结构特征为具有POU家族特有的保守结构域（POUS）和POU同源异型结构域（POUHD）,这两个结构域可与DNA上特定区域形成双向结合,进而对基因转录进行调控。Sox-2是另一种转录因子,其HMG结构域可结合在DNA的特定序列上,并可通过与Oct-4的POUs结构域之间的蛋白质．蛋白质相互作用形成POU／HMG／DNA三元复合体以调控下游靶基因的表达。文章就POU家族成员Oct-4和HMG-box家族成员Sox-2在动物早期胚胎发育中调控部分下游基因表达的分子机制进行了概述。     

转录因子Oct4、Sox2和Nanog在早期胚胎发育过程中的表达调控

胚胎发育的分子机理是现今胚胎工程及发育生物学研究迫切需要了解的。由于胚胎发育的早期阶段就是胚胎干细胞阶段,因此两者在研究上有很好的重合性。通过对调节胚胎干细胞基因转录的三个具有代表性的转录因子Oct4、Sox2和Nanog的综述,展现出胚胎发育分子机理研究概况。     

The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood

Many studies have drawn attention to the emerging role of MSC (mesenchymal stem cells) as a promising population supporting new clinical concepts in cellular therapy. However, the sources from which these cells can be isolated are still under discussion. Whereas BM (bone marrow) is presented as the main source of MSC, despite the invasive procedure related to this source, the possibility of isolating sufficient numbers of these cells from UCB (umbilical cord blood) remains controversial. Here, we present the results of experiments aimed at isolating MSC from UCB, BM and UCM (umbilical cord matrix) using different methods of isolation and various culture media that summarize the main procedures and criteria reported in the literature. Whereas isolation of MSC were successful from BM (10:10) and (UCM) (8:8), only one cord blood sample (1:15) gave rise to MSC using various culture media [DMEM (Dulbecco's modified Eagle's medium) +5% platelet lysate, DMEM+10% FBS (fetal bovine serum), DMEM+10% human UCB serum, MSCGM®] and different isolation methods [plastic adherence of total MNC (mononuclear cells), CD3⁺/CD19⁺/CD14⁺/CD38⁺‐depleted MNC and CD133⁺‐ or LNGFR⁺‐enriched MNC]. MSC from UCM and BM were able to differentiate into adipocytes, osteocytes and hepatocytes. The expansion potential was highest for MSC from UCM. The two cell populations had CD90⁺/CD73⁺/CD105⁺ phenotype with the additional expression of SSEA4 and LNGFR for BM MSC. These results clearly exclude UCB from the list of MSC sources for clinical use and propose instead UCM as a rich, non‐invasive and abundant source of MSC.     

Isolation and characterization of Oct-4+/HLA-G+ mesenchymal stem cells from human umbilical cord matrix: differentiation potential and detection of new markers

The presence of multipotent cells in several adult and embryo-related tissues opened new paths for their use in regenerative medicine. Extraembryonic tissues such as umbilical cord are considered a promising source of stem cells, potentially useful in therapy. The characterization of cells from the umbilical cord matrix (Wharton’s Jelly) and amniotic membrane revealed the presence of a population of mesenchymal-like cells, sharing a set of core-markers expressed by “mesenchymal stem cells”. Several reports enlightened the differentiation capabilities of these cells, even if at times the lack of an extensive characterization of surface markers and immune co-stimulators expression revealed hidden pitfalls when in vivo transplantation was performed. The present work describes a novel isolation protocol for obtaining mesenchymal stem cells from the umbilical cord matrix. These cells are clonogenic, retain long telomeres, can undergo several population doublings in vitro, and can be differentiated in mature mesenchymal tissues as bone and adipose. We describe for the first time that these cells, besides expressing all of the core-markers for mesenchymal stem cells, feature also the expression, at both protein and mRNA level, of tolerogenic molecules and markers of all the three main lineages, potentially important for both their differentiative potential as well as immunological features. G. La Rocca and R. Anzalone have contributed equally to this work.     

维持胚胎干细胞多能性和自我更新的转录因子Oct-4/ Nanog以及相关的调控网络

Oct-4和Nanog是两种维持干细胞多能性和自我更新的转录因子, 它们通过结合靶基因调控区, 选择性地抑制分化基因表达或促进多能性基因表达。它们通常只在多能干细胞中表达, 在分化细胞中不表达。在不同的发育阶段, 它们的表达量受到特异调控, 并且分别与Sox-2、FoxD3等其他转录因子以及LIF、BMP等胞外信号通路互相作用, 形成一个复杂的转录调节crosstalk网络, 在特异时空激活或抑制靶基因的转录; 通过互相制约最终决定干细胞是保持多能性还是分化, 以及向哪个方向分化。此外, Oct-4和Nanog对体细胞重编程为多能细胞也有重要作用。     

Sequential DNA methylation of the Nanog and Oct-4 upstream regions in human NT2 cells during neuronal differentiation

Human NT2 cells, which differentiate into neurons and astrocytes, initially express and then permanently down-regulate Nanog and Oct-4 (POU5F1). We investigated the relationship between the expression of these genes and the methylation state of their 5'-flanking regions. Gene expression and DNA methylation were assayed with quantitative polymerase chain reaction and bisulfite genomic sequencing, respectively. Retinoic acid-induced differentiation of NT2 cells to neurons is accompanied by a sequential decrease in the expression of both genes, paralleled by sequential epigenetic modification of their upstream regions. This is the first report demonstrating changes in DNA methylation in the promoter regions of Nanog and Oct-4 in a human cell line.     

Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo

Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignant transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis.     

Isolation and expansion of equine umbilical cord-derived matrix cells (EUCMCs)

Stem cells from extra-embryonic sources can be obtained by non-invasive procedures. We have standardized a method for the expansion of equine umbilical cord-derived matrix cells (EUCMCs) for potential therapy.EUCMCs were isolated from the umbilical cord of five mares immediately after delivery. For expansion, cells were grown in α-MEM and MSCBM. Moreover, to measure the effect of growth factor supplementation, epidermal growth factor (EGF) was added to α-MEM.α-MEM and MSCBM media performed similarly in terms of population doubling and CFU number value. EGF supplementation of α-MEM determined a significant increase of the population doubling value. EGF supplementation did not affect the adipogenic and chondrogenic differentiation while bone nodule sizes an increased with the osteogenic protocol.Both α-MEM and MSCBM can be used to cultivate EUCMCs. α-MEM supplemented with EGF might represent an advantage for EUCMCs expansion. The results could be useful in choosing the culture medium since α-MEM is more cost-effective than MSCBM.     

Isolation and characterization of mesenchymal stem cells from caprine umbilical cord tissue matrix

Cord tissue fills the umbilical cord around the blood vessels and contains types of stem cells (mesenchymal stem cells or MSCs) that are not generally found in cord blood. MSCs are the stem cells that give rise to many of the “support tissues” in the body, including bone, cartilage, fat and muscle. Umbilical Cord Tissue cells (UCTs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes and adipocytes have been previously isolated from different species including human, canine, murine, avian species etc. The present study documents the existence of similar multipotential stem cells in caprine UCTs having similar growth and morphological characteristics. The cells were isolated from caprine umbilical cord and cultivated in DMEM (low glucose) supplemented with 15% FBS, L-glutamine and antibiotics. Primary culture achieved confluence in 5–7 days having spindle shaped morphology. The cells were morphologically homogeneous, showed robust proliferation ability with a population doubled time of 92.07 h as well as normal karyotype. In vitro self-renewal capacity was demonstrated by colony-forming unit assay (CFU). The cells expressed MSC specific markers and showed multi-differentiation capability into adipogenic and osteogeneic. The results indicated that caprine UCTs (cUCTs) were isolated and characterized from umbilical cord tissue which can be used for tissue regeneration.     

Functional expression of the extracellular calcium sensing receptor (CaSR) in equine umbilical cord matrix size-sieved stem cells

Background

The present study investigates the effects of high external calciumconcentration ([Ca²⁺]_o) and thecalcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, ongrowth/proliferation of two equine size-sieved umbilical cord matrixmesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observedcell response was analyzed at both the mRNA and protein level.

Methodology/Principal Findings

A large (>8 µm in diameter) and a small (<8 µm) cell linewere cultured in medium containing: 1) low[Ca²⁺]_o (0.37 mM); 2) high[Ca²⁺]_o (2.87 mM); 3) NPS R-467 (3µM) in presence of high [Ca²⁺]_oand 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed byincubation in presence of NPS R-467 in medium with high[Ca²⁺]_o. Growth/proliferation rateswere compared between groups. In large cells, the addition of NPS R-467significantly increased cell growth whereas increasing[Ca²⁺]_o was not effective in thiscell line. In small cells, both higher[Ca²⁺]_o and NPS R-467 increasedcell growth. In both cell lines, preincubation with the CaSR antagonist NPS2390 significantly inhibited the agonistic effect of NPS R-467. In both celllines, increased [Ca²⁺]_o and/or NPSR-467 reduced doubling time values.Treatment with NPS R-467 down-regulatedCaSR mRNA expression in both cell lines. In large cells, NPS R-467 reducedCaSR labeling in the cytosol and increased it at cortical level.

Conclusions/Significance

In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNAexpression and stimulate cell growth/proliferation in eUCM-MSC. Their use ascomponents of media for eUCM-MSC culture could be beneficial to obtainenough cells for down-stream purposes.