Prolonged Hypoxia Concomitant with Serum Deprivation Induces Massive Human Mesenchymal Stem Cell Death (original) (raw)
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Effects of Oxygen and Glucose on Bone Marrow Mesenchymal Stem Cell Culture
Advanced Biosystems, 2020
including skin, bone, cartilage, heart tissues, blood vessels, etc., have successfully been recreated in vitro, [3-5] their clinical application is limited. Most tissues are highly vulnerable to ischemia, [6] hindering the survival of clinically relevant tissue volumes after implantation in vivo of engineered constructs. The inability to deliver oxygen and nutrients to cells within scaffolds is thought to result in cell death and lack of efficiency of transplanted cells. [7-9] Indeed, oxygen and nutrient supply in bioreactors is only possible through the movement of the culture medium through their structure and is limited within the tissue by diffusion. [10] Furthermore, such constructs do not possess a functioning blood vessel network that could anastomose to the host vasculature upon implantation. Without an integrated vascularization, their in vivo survival depends on the host's ability to vascularize the graft and previous research has shown that the complete vascularization of a 3 mm tissue engineered construct could take between one and two weeks. [11,12] During this time, cells embedded deep within these constructs become hypoxic and in some cases necrotic. [7,9] This also hampers important cellular functions such as cell proliferation and neo-vascularization. [13] Mesenchymal stem cells (MSCs) hold great potential in tissue engineering application due mainly to the beneficial factors they secrete and to their stem cell properties. Some of these include proangiogenic and immunosuppressive functions, the ability to differentiate into multiple cell lineages, and the ability to interact and regulate cells within both the innate and adaptive immune systems. [14-19] Through cell to cell communication, they also help mediate release of specific growth factors and cytokines that promote cell survival. [20-25] In vitro MSC are routinely cultured in "normoxia," i.e., 20% oxygen, namely atmospheric air and 5% CO 2 , but the physoxic levels are estimated to be 3-6% for human stem cell niches and many studies have indicated differences in behavior when cultured in normoxia, physoxia, and hypoxia. We sought to determine whether MSC could be cultured in the absence of oxygen, and whether glucose availability affected long term anoxic survival (Figure 1). Erythropoietin (EPO) producing MSC allowed us to measure protein production and also to determine if EPO, a protein reported to improve anoxic survival preclinically, [26] could have any effect on anoxic MSC survival. This study determines whether the viability of mesenchymal stem cell (MSC) in vitro is most sensitive to oxygen supply, energetic substrate supply, or accumulation of lactate. Mouse unmodified (wild type (WT)) and erythropoietin (EPO) gene-modified MSC is cultured for 7 days in normoxic (21%) and anoxic conditions. WT-MSC is cultured in anoxia for 45 days in high and regular glucose media and both have similar viability when compared to their normoxic controls at 7 days. Protein production of EPO-MSC is unaffected by the absence of oxygen. MSC doubling time and post-anoxic exposure is increased (WT: 32.3-73.3 h; EPO: 27.2-115 h). High glucose leads to a 37% increase in cell viability at 13 days and 17% at 30 days, indicating that MSC anoxic survival is affected by supply of metabolic substrate. However, after 30 days, little difference in viability is found, and at 45 days, complete cell death occurs in both the conditions. This death cannot be attributed to lack of glucose or lactate levels. MSC stemness is retained for both osteogenic and adipogenic differentiations. The absence of oxygen increases the doubling time of MSC but does not affect their viability, protein production, or differentiation capacity.
Journal of Cellular and Molecular Medicine, 2011
The present study examined the respective roles of glucose and continuous severe hypoxia on MSC viability and function with respect to bone tissue engineering. We hereby demonstrate for the first time that MSCs survive exposure to long-term (12 days), severe (pO2 Ͻ 1.5 mmHg) hypoxia, provided glucose is available. To this end, an in vitro model that mimics the hypoxic environment and cell-driven metabolic changes encountered by grafted sheep cells was established. In this model, the hallmarks of hypoxia (low pO2, hypoxia inducible factor-1␣ expression and anaerobic metabolism) were present. When conditions switched from hypoxic (low pO2) to ischemic (low pO2 and glucose depletion), MSCs exhibited shrinking, decreased cell viability and ATP content due to complete exhaustion of glucose at day 6; these results provided evidence that ischemia led to the observed massive cell death. Moreover, MSCs exposed to severe, continuous hypoxia, but without any glucose shortage, remained viable and maintained both their in vitro proliferative ability after simulation with blood reperfusion at day 12 and their in vivo osteogenic ability. These findings challenge the traditional view according to which severe hypoxia per se is responsible for the massive MSC death observed upon transplantation of these cells and provide evidence that MSCs are able to withstand exposure to severe, continuous hypoxia provided that a glucose supply is available.
Journal of Cellular Physiology, 2009
The low bone marrow (BM) MSC titers demand a fast ex vivo expansion process to meet the clinically relevant cell dosage. Attending to the low oxygen tension of BM in vivo, we studied the influence of hypoxia on human BM MSC proliferation kinetics and metabolism. Human BM MSC cultured under 2% (hypoxia) and 20% O2 (normoxia) were characterized in terms of proliferation, cell division kinetics and metabolic patterns. BM MSC cultures under hypoxia displayed an early start of the exponential growth phase, and cell numbers obtained at each time point throughout culture were consistently higher under low O2, resulting in a higher fold increase after 12 days under hypoxia (40 ± 10 vs. 30 ± 6). Cell labeling with PKH26 allowed us to determine that after 2 days of culture, a significant higher cell number was already actively dividing under 2% compared to 20% O2 and BM MSC expanded under low oxygen tension displayed consistently higher percentages of cells in the latest generations (generations 4–6) until the 5th day of culture. Cells under low O2 presented higher specific consumption of nutrients, especially early in culture, but with lower specific production of inhibitory metabolites. Moreover, 2% O2 favored CFU-F expansion, while maintaining BM MSC characteristic immunophenotype and differentiative potential. Our results demonstrated a more efficient BM MSC expansion at 2% O2, compared to normoxic conditions, associated to an earlier start of cellular division and supported by an increase in cellular metabolism efficiency towards the maximization of cell yield for application in clinical settings. J. Cell. Physiol. 223: 27–35, 2010. © 2009 Wiley-Liss, Inc.
Hypoxic Culture Conditions as a Solution for Mesenchymal Stem Cell Based Regenerative Therapy
The Scientific World Journal, 2013
Cell-based regenerative therapies, based on in vitro propagation of stem cells, offer tremendous hope to many individuals suffering from degenerative diseases that were previously deemed untreatable. Due to the self-renewal capacity, multilineage potential, and immunosuppressive property, mesenchymal stem cells (MSCs) are considered as an attractive source of stem cells for regenerative therapies. However, poor growth kinetics, early senescence, and genetic instability during in vitro expansion and poor engraftment after transplantation are considered to be among the major disadvantages of MSC-based regenerative therapies. A number of complex inter-and intracellular interactive signaling systems control growth, multiplication, and differentiation of MSCs in their niche. Common laboratory conditions for stem cell culture involve ambient O 2 concentration (20%) in contrast to their niche where they usually reside in 2-9% O 2 . Notably, O 2 plays an important role in maintaining stem cell fate in terms of proliferation and differentiation, by regulating hypoxia-inducible factor-1 (HIF-1) mediated expression of different genes. This paper aims to describe and compare the role of normoxia (20% O 2 ) and hypoxia (2-9% O 2 ) on the biology of MSCs. Finally it is concluded that a hypoxic environment can greatly improve growth kinetics, genetic stability, and expression of chemokine receptors during in vitro expansion and eventually can increase efficiency of MSC-based regenerative therapies.
Stem Cells, 2019
After in vivo transplantation, mesenchymal stem cells (MSC) face an ischemic microenvironment, characterized by nutrient deprivation and reduced oxygen tension, which reduces their viability and thus their therapeutic potential. Therefore, MSC response to models of in vitro ischemia is of relevance for improving their survival and therapeutic efficacy. The aim of this study was to understand the survival/adaptive response mechanism that MSC use to respond to extreme culture conditions. Specifically, the effect of a long-term starvation on human bone marrow (hBM)-derived MSC cultured in a chemically defined medium (fetal bovine serum-free [SF] and human SF), either in hypoxic or normoxic conditions. We observed that hBM-MSC that were isolated and cultured in SF medium and subjected to a complete starvation for up to 75 days transiently changed their behavior and phenotype. However, at the end of that period, hBM-MSC retained their characteristics as determined by their morphology, DN...
Cell Transplantation
Embryonic stem cells (ESCs) have the potential to be used as an unlimited cell source for cell transplantation therapy, as well as for studying mechanisms of disease and early mammalian development. However, applications involving ESCs have been limited by the lack of reliable differentiation methods in many cases. Mesenchymal stem cells (MSCs) have also emerged as a promising cell source, but as suggested in recent studies, these cells display limited potential for proliferation and differentiation, thereby limiting their usefulness in the clinic and in the laboratory. Unfortunately, effective methods for induction of MSCs from pluripotent stem cells have not been established, and the development of such methods remains a major challenge facing stem cell biologists. Oxygen concentration is one of the most important factors regulating tissue development. It has profound effects on cell metabolism and physiology, and can strongly influence stem cell fate. Here we demonstrate that severe-low O ₂ concentrations (1%) can function as a selective pressure for removing undifferentiated pluripotent cells during the induction of MSCs from rabbit ESCs (rESCs), and that MSCs induced under severe hypoxic conditions function as normal MSCs; i.e., they repopulate after cloning, express specific markers (Vimentin, CD29, CD90, CD105 and CD140a) and differentiate into adipocytes, osteoblasts and chondrocytes. Furthermore, we demonstrate that these cells can contribute to cartilage regeneration in an in vivo rabbit model for joint cartilage injury. These results support the notion that exposing ESCs to severe hypoxic conditions during differentiation can be used as a strategy for the preparation of functional MSCs from ESCs.
Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells
Stem cell research & therapy, 2018
In the bone marrow, MSCs reside in a hypoxic milieu (1-5% O) that is thought to preserve their multipotent state. Typically, in vitro expansion of MSCs is performed under normoxia (~ 21% O), a process that has been shown to impair their function. Here, we evaluated the characteristics and function of MSCs cultured under hypoxia and hypothesized that, when compared to normoxia, dedicated hypoxia will augment the functional characteristics of MSCs. Human and porcine bone marrow MSCs were obtained from fresh mononuclear cells. The first study evaluated MSC function following both long-term (10 days) and short-term (48 h) hypoxia (1% O) culture. In our second study, we evaluated the functional characteristics of MSC cultured under short-term 2% and 5% hypoxia. MSCs were evaluated for their metabolic activity, proliferation, viability, clonogenicity, gene expression, and secretory capacity. In long-term culture, common MSC surface marker expression (CD44 and CD105) dropped under hypoxia....
Optimization of Pre-transplantation Conditions to Enhance the Efficacy of Mesenchymal Stem Cells
International Journal of Biological Sciences, 2015
Mesenchymal stem cells (MSCs) are considered a potential tool for cell based regenerative therapy due to their immunomodulatory property, differentiation potentials, trophic activity as well as large donor pool. Poor engraftment and short term survival of transplanted MSCs are recognized as major limitations which were linked to early cellular ageing, loss of chemokine markers during ex vivo expansion, and hyper-immunogenicity to xeno-contaminated MSCs. These problems can be minimized by ex vivo expansion of MSCs in hypoxic culture condition using well defined or xeno-free media i.e., media supplemented with growth factors, human serum or platelet lysate. In addition to ex vivo expansion in hypoxic culture condition using well defined media, this review article describes the potentials of transient adaptation of expanded MSCs in autologous serum supplemented medium prior to transplantation for long term regenerative benefits. Such transient adaptation in autologous serum supplemented medium may help to increase chemokine receptor expression and tissue specific differentiation of ex vivo expanded MSCs, thus would provide long term regenerative benefits.
Cytotechnology, 2014
Mesenchymal stem cells (MSCs) are accepted as a promising tool for therapeutic purposes. However, low proliferation and early senescence are still main obstacles of MSCs expansion for using as cell-based therapy. Thus, clinical scale of cell expansion is needed to obtain a large number of cells serving for further applications. In this study, we investigated the value of embryonic stem cells conditioned medium (ESCM) for in vitro expansion of Wharton's jellyderived mesenchymal stem cells (WJ-MSCs) as compared to typical culture medium for MSCs, Dulbecco's modified Eagle's medium with 1.0 g/l glucose (DMEM-LG) supplemented with 10 % FBS, under hypoxic condition. The expanded cells from ESCM (ESCM-MSCs) and DMEM-LG (DMEM-MSCs) were characterized for both phenotype and biological activities including proliferation rate, population doubling time, cell cycle distribution and MSCs characteristics. ESCM and DMEM-LG could enhance WJ-MSCs proliferation as 204.66 ± 10.39 and 113.77 ± 7.89 fold increase at day 12, respectively. ESCM-MSCs could express pluripotency genes including Oct-4, Oct-3/4, Nanog, Klf-4, C-Myc and Sox-2 both in early and late passages whereas the downregulations of Oct-4 and Nanog were detected in late passage cells of DMEM-MSCs. The 2 cell populations also showed common MSCs characteristics including normal cell cycle, fibroblastic morphology, cell surface markers expressions (CD29 ? , CD44 ? , CD90 ? , CD34-, CD45-) and differentiation capacities into adipogenic, chondrogenic and osteogenic lineages. Moreover, our results revealed that ESCM exhibited as a rich source of several factors which are required for supportive WJ-MSCs proliferation. In conclusion, ESCM under hypoxic condition could accelerate WJ-MSCs expansion while maintaining their pluripotency properties. Our knowledge provide short term and cost-saving in WJ-MSCs expansion which has benefit to overcome insufficient cell numbers for clinical applications by reusing the discarded cell culture supernates from human ES culture system. Moreover, these findings can also apply for stem cell banking, regenerative medicine and pharmacological applications.