Hypoxia enhances the viability,growth and chondrogenic potential of cryopreserved human adipose-derived stem cells

Cryopreservation is the only existing method of storage of human adipose-derived stem cells (ASCs) for clinical use. However, cryopreservation has been shown to be detrimental to ASCs, particularly in term of cell viability. To restore the viability of cryopreserved ASCs, it is proposed to culture the cells in a hypoxic condition. To this end, we aim to investigate the effect of hypoxia on the cryopreserved human ASCs in terms of not only cell viability, but also their growth and stemness properties, which have not been explored yet. In this study, human ASCs were cultured under four different conditions: fresh (non-cryopreserved) cells cultured in 1) normoxia (21% O₂) and 2) hypoxia (2% O₂) and cryopreserved cells cultured in 3) normoxia and 4) hypoxia. ASCs at passage 3 were subjected to assessment of viability, proliferation, differentiation, and expression of stemness markers and hypoxia-inducible factor-1 alpha (HIF-1α). We found that hypoxia enhances the viability and the proliferation rate of cryopreserved ASCs. Further, hypoxia upregulates HIF-1α in cryopreserved ASCs, which in turn activates chondrogenic genes to promote chondrogenic differentiation. In conclusion, hypoxic-preconditioned cryopreserved ASCs could be an ideal cell source for cartilage repair and regeneration.     

Effect of low oxygen tension on the biological characteristics of human bone marrow mesenchymal stem cells

Culture of mesenchymal stem cells (MSCs) under ambient conditions does not replicate the low oxygen environment of normal physiological or pathological states and can result in cellular impairment during culture. To overcome these limitations, we explored the effect of hypoxia (1 % O₂) on the biological characteristics of MSCs over the course of different culture periods. The following biological characteristics were examined in human bone marrow-derived MSCs cultured under hypoxia for 8 weeks: proliferation rate, morphology, cell size, senescence, immunophenotypic characteristics, and the expression levels of stemness-associated factors and cytokine and chemokine genes. MSCs cultured under hypoxia for approximately 2 weeks showed increased proliferation and viability. During long-term culture, hypoxia delayed phenotypic changes in MSCs, such as increased cell volume, altered morphology, and the expression of senescence-associated-β-gal, without altering their characteristic immunophenotypic characteristics. Furthermore, hypoxia increased the expression of stemness and chemokine-related genes, including OCT4 and CXCR7, and did not decrease the expression of KLF4, C-MYC, CCL2, CXCL9, CXCL10, and CXCR4 compared with levels in cells cultured under normoxia. In conclusion, low oxygen tension improved the biological characteristics of MSCs during ex vivo expansion. These data suggest that hypoxic culture could be a useful method for increasing the efficacy of MSC cell therapies.     

Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O₂) environment, whereas, their in-vitro expansion is typically performed under normoxia (20–21% O₂). The comparative information about the effects of low and normal O₂ levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O₂) and normoxia (21% O₂). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p?<?0.05) improved, thus, yielding a significantly (p?<?0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O₂ conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p?<?0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

     

Increased Proliferation and Analysis of Differential Gene Expression in Human Wharton's Jelly-derived Mesenchymal Stromal Cells under Hypoxia

Multipotent mesenchymal stromal cells (MSCs) from Wharton''s jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.     

In Situ Normoxia Enhances Survival and Proliferation Rate of Human Adipose Tissue-Derived Stromal Cells without Increasing the Risk of Tumourigenesis

Adipose tissue-derived stromal cells (ASCs) natively reside in a relatively low-oxygen tension (i.e., hypoxic) microenvironment in human body. Low oxygen tension (i.e., in situ normoxia), has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O₂ concentration (21% O₂) or in situ normoxia (2% O₂). We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O₂ concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O₂ as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.     

Basal medium composition and serum or serum replacement concentration influences on the maintenance of murine embryonic stem cells

The expansion of stem cell numbers while retaining their developmental properties is a bioprocess challenge. We compared the growth rates and embryoid body (EB) formation yields of R1 and EFC murine embryonic stem cells (mESC) cultured in two basal media (DMEM or DMEM:F12) with additions of 1.7–15% fetal bovine serum (FBS) or serum replacer (KOSR). Whereas the basal medium or KOSR dose did not have a significant effect on growth rate for either cell line, increasing doses of KOSR had a significant negative effect on the EB yield of EFC cells. Use of DMEM:F12 and increasing doses of FBS independently and significantly increased the growth rate for both cell lines. DMEM:F12 also significantly increased EB yields for both cell lines. The results show that use of DMEM:F12 and several-fold lower than conventional concentrations of KOSR can efficiently support maintenance of mESC and that KOSR should be dose as well as lot optimized.     

Hypoxia enhances buffalo adipose-derived mesenchymal stem cells proliferation,stemness, and reprogramming into induced pluripotent stem cells

Adipose tissue-derived mesenchymal stem cells (ASCs) from livestock are valuable resources for animal reproduction and veterinary therapeutics. Previous studies have shown that hypoxic conditions were beneficial in maintaining the physiological activities of ASCs. However, the effects of hypoxia on buffalo ASCs (bASCs) remain unclear. In this study, the effects of hypoxia on proliferation, stemness, and reprogramming into induced pluripotent stem cells (iPSCs) of bASCs were examined. The results showed that the hypoxic culture conditions (5% oxygen) enhanced the proliferation and colony formation of bASCs. The expression levels of proliferation-related genes, and secretion of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were significantly enhanced in hypoxia. Hypoxic culture conditions activated hypoxia-inducible factor-1α (HIF-1α), thereby contributing to the secretion of bFGF and VEGF, which in turn enhanced the expression of HIF-1α and promoted the proliferation of bASCs. Furthermore, in hypoxic culture conditions, bASCs exhibited the main characteristics of mesenchymal stem cells, and the expression levels of the pluripotent markers OCT4, NANOG, C-MYC, and the differentiation capacity of bASCs were significantly enhanced. Finally, bASCs were more efficiently and easily reprogrammed into iPSCs in hypoxic culture conditions and these iPSCs exhibited some characteristics of naïve pluripotent stem cells. These findings provide the theoretical guidance for elucidating the detailed mechanism of hypoxia on physiological activities of bASCs including proliferation, stemness maintenance, and reprogramming.     

Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue

ABSTRACT: BACKGROUND: Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2. RESULTS: At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O2 for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state. CONCLUSIONS: Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.     

胚胎干细胞向心肌细胞分化过程中能量代谢变化的研究

目的：定向诱导人胚胎干细胞分化为心肌细胞，对分化过程中胚胎干细胞、心肌祖细胞和心肌细胞糖酵解能力和线粒体氧化磷酸化能力进行实时定量检测，探索分化过程中细胞能量代谢表型的转换机制.方法：用GSK3抑制剂CHIR99021和Wnt信号通路小分子抑制剂IWP2的方法定向分化人胚胎干细胞为心肌祖细胞和心肌细胞；细胞免疫荧光检测人胚胎干细胞标志物，流式细胞术检测人心肌祖细胞和心肌细胞标志物；应用细胞外流量分析（Extracellular Flux Analysis）方法检测人胚胎干细胞、心肌祖细胞和心肌细胞能量代谢情况.结果：人胚胎干细胞干性保持稳定，均表达Nanog、OCT4、SOX2细胞标志物；在向心肌分化过程中，第7d心肌祖细胞标志物Isl1表达99%以上，分化第15d心肌细胞标志物cTnT表达83%以上；人胚胎干细胞糖酵解代谢能力最强，心肌细胞线粒体功能最强，心肌祖细胞处于两种代谢方式的过度阶段.结论：在人胚胎干细胞向心肌细胞分化的过程中，细胞糖酵解能力逐渐减弱，线粒体氧化磷酸化能力逐渐增强，细胞的能量代谢类型发生转变.     

Human Retinal Progenitor Cell Transplantation Preserves Vision

Cell transplantation is a potential therapeutic strategy for retinal degenerative diseases involving the loss of photoreceptors. However, it faces challenges to clinical translation due to safety concerns and a limited supply of cells. Human retinal progenitor cells (hRPCs) from fetal neural retina are expandable in vitro and maintain an undifferentiated state. This study aimed to investigate the therapeutic potential of hRPCs transplanted into a Royal College of Surgeons (RCS) rat model of retinal degeneration. At 12 weeks, optokinetic response showed that hRPC-grafted eyes had significantly superior visual acuity compared with vehicle-treated eyes. Histological evaluation of outer nuclear layer (ONL) characteristics such as ONL thickness, spread distance, and cell count demonstrated a significantly greater preservation of the ONL in hRPC-treated eyes compared with both vehicle-treated and control eyes. The transplanted hRPCs arrested visual decline over time in the RCS rat and rescued retinal morphology, demonstrating their potential as a therapy for retinal diseases. We suggest that the preservation of visual acuity was likely achieved through host photoreceptor rescue. We found that hRPC transplantation into the subretinal space of RCS rats was well tolerated, with no adverse effects such as tumor formation noted at 12 weeks after treatment.     

Retinal stem/progenitor properties of iris pigment epithelial cells

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina and ciliary epithelium of the adult eye. Here, we first demonstrate the characterization of neural stem/progenitors in postnatal iris pigment epithelial (IPE) cells. Pure isolated IPE cells could form spheres that contained cells expressing retinal progenitor markers in non-adherent culture. The spheres grew by cell proliferation, as indicated by bromodeoxyuridine incorporation. When attached to laminin, the spheres forming IPE derived cells were able to exhibit neural phenotypes, including retinal-specific neurons. When co-cultured with embryonic retinal cells, or grafted into embryonic retina in vivo, the IPE cells could also display the phenotypes of photoreceptor neurons and Muller glia. Our results suggest that the IPE derived cells have retinal stem/progenitor properties and neurogenic potential without gene transfer, thereby providing a novel potential source for both basic stem cell biology and therapeutic applications for retinal diseases.     

Biochemical and morphological effects of hypoxic environment on human embryonic stem cells in long-term culture and differentiating embryoid bodies

The mammalian reproductive tract is known to contain 1.5–5.3% oxygen (O₂), but human embryonic stem cells (hESCs) derived from preimplantation embryos are typically cultured under 21% O₂ tension. The aim of this study was to investigate the effects of O₂ tension on the long-term culture of hESCs and on cell-fate determination during early differentiation. hESCs and embryoid bodies (EBs) were grown under different O₂ tensions (3, 12, and 21% O₂). The expression of markers associated with pluripotency, embryonic germ layers, and hypoxia was analyzed using RTPCR, immunostaining, and Western blotting. Proliferation, apoptosis, and chromosomal aberrations were examined using BrdU incorporation, caspase-3 immunostaining, and karyotype analysis, respectively. Structural and morphological changes of EBs under different O₂ tensions were comparatively examined using azan- and hematoxylineosin staining, and scanning and transmission electron microscopy. Mild hypoxia (12% O₂) increased the number of cells expressing Oct4/Nanog and reduced BrdU incorporation and aneuploidy. The percentage of cells positive for active caspase-3, which was high during normoxia (21% O₂), gradually decreased when hESCs were continuously cultured under mild hypoxia. EBs subjected to hypoxia (3% O₂) exhibited well-differentiated microvilli on their surface, secreted high levels of collagen, and showed enhanced differentiation into primitive endoderm. These changes were associated with increased expression of Foxa2, Sox17, AFP, and GATA4 on the EB periphery. Our data suggest that mild hypoxia facilitates the slow mitotic division of hESCs in long-term culture and reduces the frequency of chromosomal abnormalities and apoptosis. In addition, hypoxia promotes the differentiation of EBs into extraembryonic endoderm.     

RPE-derived factors modulate photoreceptor differentiation: A possible role in the retinal stem cell niche

A photoreceptor cell line, designated 661W, was tested for its response to growth factors secreted by retinal pigment epithelial cells including basic fibroblast growth factor, epidermal growth factor, and nerve growth factor. Early passaged 661W cells expressed high levels of retinal progenitor markers such as nestin and Pax6, but not opsin or glial fibrillary acidic protein. 661W cells grown in FGF-2 or EGF exhibited a multiple-process morphology with small phase-bright nuclei similar to neurons, whereas cells cultured in nerve growth factor (NGF) or retinal pigment epithelium (RPE)-conditioned medium (RPE-CM) displayed rounded profiles lacking processes. 661W cells grown in FGF-2 were slightly elevated, but not significantly above, control cultures; but cells treated with RPE-CM or NGF were fewer, ∼63% and 49% of control, respectively. NGF immunodepletion of RPE-CM strongly suppressed the inhibitory activity of RPE-CM on cell proliferation. Cells treated with FGF-2, but not NGF, upregulated their expression of opsin. All treatment conditions resulted in almost 100% viability based on calcium AM staining. Cells grown on extracellular matrix proteins laminin, fibronectin, and/or collagen resembled those grown on untreated dishes. This study showed that early passaged 661W cells displayed characteristics of retinal progenitor cells. The 661W cells proliferated and appeared to mature morphologically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited proliferation and morphological differentiation of 661W cells, possibly inducing cell cycle arrest. These findings are consistent with reports that the RPE modulates photoreceptor differentiation and retinal progenitor cells via secreted factors and may play a role in the regulation of the retinal stem cell niche.     

Mesenchymal stromal progenitor cells: general characteristics and functional state in low oxygen tension

The promising use of multipotent mesenchymal stromal cells (MSC) in regenerative technologies accounts for necessity of detailed study and analysis of their physiology. Along with searching for MSC specific immunophenotype markers and factors that affect MSC heterogeneity, proliferative activity, self-renewal capacity and differentiation potential, progenitor, the cells functioning in low oxygen tension present one the open-ended problems. The specific response of progenitor cells to hypoxia seems to depend on many factors including their commitment and differentiation status, tissue type as well as the nature of the hypoxic impact. Interestingly, it was shown that low oxygen tension can be used as a factor regulating MSC proliferation, viability and differentiation.     

缺氧对新生小牛肺血管平滑肌细胞增殖的影响及764—3的抑制作用

本研究应用细胞培养、~3H—TdR参入技术,探讨缺氧对新生小牛肺血管平滑肌细胞(PASMC)DNA合成和细胞增殖的影响及746—3和川芎嗪抗细胞增殖效果。结果表明:缺氧的肺血管内皮细胞(PAEC)培养液可以明显增加PASMC中~3H—TdR的参入;缺氧24h亦可直接刺激PASMC,使其中~3H—TdR的参入显著增加(P<0.001);在缺氧的PASMC培养基中加入764—3,~3H—TdR参入值与单纯缺氧组相比显著下降,并使细胞数减少36.3％,764—3还可以显著抑制常氧PASMC的增殖;川芎嗪的作用较小。实验结果提示,缺氧不仅可以刺激内皮细胞(EC)产生促分裂素使PASMC增殖,而且还可以直接刺激PASMC的增殖,764—3可抑制缺氧及血清刺激的PASMC的增殖。     

低氧通过上调Wnt5a促进人胚胎干细胞向生血内皮细胞分化

胚胎的早期发育是在低氧条件下进行的,低氧环境在胚胎血管发生及造血发育中起着重要作用,低氧条件能促进胚胎干细胞在体外向内皮细胞和造血细胞的分化,但低氧条件对造血细胞产生的具体作用及相应机制尚不清楚．本研究利用人Es细胞向造血祖细胞定向分化体系,发现低氧环境可以促进CD31＋TIE2＋造血内皮祖细胞的产生,2天后造血内皮祖细胞开始表达终生造血基因．进一步研究发现,低氧能够上调Wnt5a的表达,干涉Wnt5a能够抑制低氧环境对生血内皮细胞分化的促进作用．在正常氧环境下加入Wnt5a产生促进生血内皮细胞分化的效应,该效应与低氧处理促进生血内皮细胞产生的作用相似．本研究首次证明了低氧通过上调Wnt5a的表达促进人Es细胞向生血内皮细胞的分化,为ES细胞向生血内皮细胞的分化及造血祖细胞分化的研究提供了新的线索．     

Effects of hypoxia on proliferation of human cord blood-derived mesenchymal stem cells

The purpose of our study was to examine the influence of hypoxia on proliferation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). The mononuclear cells were separated by density gradient centrifugation from human umbilical cord blood and then, respectively, cultured under hypoxia (5 % O₂) or normoxia (20 % O₂). Their cell morphology, cell surface markers, β-galactosidase staining, cell growth curve, DNA cycle, and the expression of hypoxia-inducible factor-1α (HIF-1α) were evaluated. We found that hypoxia, in part via HIF-1α, improved the proliferation efficiency, and prevented senescence of hUCB-MSCs without altering their morphology and surface markers. These results demonstrated that hypoxia provides a favorable culture condition to promote hUCB-MSCs proliferation in vitro, which is a better way to obtain sufficient numbers of hUCB-MSCs for research and certainly clinical application.