Effect Of Lactic Acid On The Proliferation And Osteogenic Differentiation Of Amniotic Mesenchymal Stem Cells In Vitro

In comparison with the most widely explored bone marrow-derived mesenchymal stem cells (BMMSCs), amniotic MSCs (hAMSCs) are readily available and easily procured without invasive procedures, and display immunomodulatory properties and no ethical concerns. Due to these advantages, hAMSCs represent an attractive candidate for tissue regeneration, cell therapy and gene therapy. However, hAMSCs are present in small quantities and mainly dormant in tissues and previous studies have shown that hAMSCs tend to lose their proliferative capacity and differentiation potential during in vitro expansion. Therefore, their clinical application is limited as a result of both insufficient cell quantity and low quality. Proliferation and differentiation of hAMSCs can be affected by the changes in their local environment such as nutritional supply and accumualation of waste products. Lactic acid, a major waste product, is mainly produced from glucose metabolism, and can also be generated from other sources such as biomaterials including poly (lactic acid) (PLA) and poly (lactic-co-glycolic acid) (PLGA). Previous study has shown that lactic acid could inhibit cell proliferation and glucose metabolism, and influence antibody production of animal cells and the differentiation of stem cells. However, the molecular mechanisms underlying this inhibitory effect of lactic acid on the proliferation and osteogenic differentiation of hAMSCs are not well understood yet. In addition, conventional static culture methods, generally performed in culture flasks or plates with flat surfaces, have limited cell growth area and need frequent cell passaging based on enzyme digestion. Moreoever, the culture environment is heterogeneous in nature due to the lack of fluid mixing. Therefore, by using conventional static culture methods, it is hard to scale up the expansion and induce lineage-specific differention and the cell products are lack of consistency, which can not meet the needs of quantity and quality and controlled differention in clinical applications. So it is important to understand the effect of culture environment on the growth, metabolism, and differention of hAMSCs.First, this study investigated the effects of normoxia (20% O2) and hypoxia (5% O2) on the expansion, metabolism and osteogenic differentiation potential of hAMSCs and BMMSCs. Compared to normal oxygen, hypoxia promoted the growth of both hAMSCs and BMMSCs. However, hypoxia was not favorable for the osteogenic differentiation of hAMSCs, which often led to the cell death and detatchment. Notably, the production of lactic acid in growth medium increased significantly under hypoxia, expecially during the later period, in which the concentration of lactic acid exceeded 15 mmol/L. The specific lactic acid production rate and the lactic acid yield coefficient from glucose increased by about 89% and 45% over these cultured under normoxia, respectively. In addition, when hAMSCs were induced in osteogenic medium, the concentration of lactic acid exceeded 15 mmol/L even under normoxia.Second, due to the drastic accumulation of lactic acid during the growth and osteogenic induction, the effect of lactic acid on the growth, metabolism, colony-forming capacity, and osteogenic differentiation of hAMSCs were investigated. It was shown that the growth, metabolism, and colony-forming capacity of hAMSCs were inhibited by lactic acid at a concentration higher than 10 mmol/L. This influence was attributed to both decreased pH and the chemical action of lactate ion, but the osmotic pressure had little contribution. In addition, lactic acid could inhibit osteogenic differentiaion of hAMSCs with the concentration above 20 mmol/L. This was because that decreased pH and the chemical action of lactate ion could decrease the ALP activity and osteopontin expression by inhibiting the expression of Osterix gene, which eventually resulted in the reduction of bone matrix mineralization. Further research showed that the inhibition of lactic acid on osteogenic differentiation might also be related to the observed increase in adipogenic differentiation. Similarly, the lactic acid also inhibited the osteogenic differentiation of BMMSCs. But, this influence was not only resulted from the chemical action of lactate ion, but also the decreased pH as well as high osmotic pressure (334 mOsm/kg). However, for cells induced in osteogenic medium, lactic acid induced a significant increase in the growth of hAMSCs and BMMSCs, which was in contrast with the significant inhibition on cell growth in growth medium.In the end, based on the above study on the effect of lactic acid on the growth and osteogenic differentiation of hAMSCs, a microcarrier supension culture system in bioreactor was explored to implement the large-scale expansion and osteogenic induction of hAMSCs. The effects of oxygen and medium exchange on the maintaince of stem cell gene expression and osteogenic differentiation capacity of hAMSCs after expansion in bioreactor were investigated. In addition, the osteogenic characteristics of hAMSCs in bioreactor were also studied with those under the conventional static induction as control. The results showed that the microcarrier supension culture system in bioreactor promoted the cell growth. Compared to the conventional static culture under normoxia with a maximum cell density of 2.13×105 cells/ml, the maximum cell density in bioreactor could reach 6.88×105 cells/ml. In addition, the cell expansion, the stem cell gene-Oct-4 and Nanog expression, and the osteogenic capacity of hAMSCs cultured under hypoxia were significantly enhanced in comparison with those achieved in bioreactor under normoxia. However, more lactic acid was accumulated when cultured under hypoxia. The lactic acid-reducing cultivation protocol with medium exchange everyday was more favorable for cell growth and the expression of Oct-4 gene than that with medium change every 1.5 d and however had little effect on the maintaince of osteogenic capacity. Strikingly, in contrast with the conventional static induction, the expression of early osteoblast transcription factors Runx2 and Osterix was upregulated in cells cultured in the microcarrier supension induction system. In accordance with these, the gene expression of type I collagen, osteopontin and osteocalcin as well as the ALP activity and bone matrix mineralization were also enhanced.Taken together, these findings obtained from this work provide valuable knowledge on the mechanism of lactic acid influencing the proliferation and osteogenic differention of hAMSCs. And, a scalable microcarrier-based bioreactor culture system for both expansion and osteogenic induction of hAMSCs were explored. Our resutls also provide a basis for better fundamental understanding of the relationship between the physiological characteristics of stem cells and the culture conditions. Further, new perspectives on the regulation of in vivo microenviroment on the growth capacity and differentiation potential of hAMSCs may also be inspired. Nevertheless, this work offers novel insights into optimizing culture parameters and developing bioreactors for stem cell research in the future.