Abstract
There is an increasing trend in the use of Chinese herbs for medical usage as well as for the development of stem cells for an alternative medical treatment for illnesses . This article investigated the biological effects of the popular Chinese herb called Astragalus membranaceus on the umbilical cord mesenchymal stem cell (UCMSC). The two main objectives are to determine the promotion of proliferation and immunosuppression of UCMSC.
Astragalus membranaceus has a biological function of controlling the vital energy deficiency, it was hypothesized to improve on the immunosuppressive properties of UCMSC. To carry out this study, Astragalus polysaccharides (APS) which was extracted from Astragalus membranaceus and added to the culture medium of UCMSC in the form of PG2 injection. Immunosuppressive effects and other biological functions such as proliferation were compared against the control samples. Positive results were obtained for the effects of PG2 on the UCMSC proliferation and their immunosuppressive effects which would encouraging for further development as UCMSC could be a more effective immune disorder treatment.
The current limitation of the study is that APS is only tested to be applicable to the one type of stem cells which is the UCMSCs. Furthermore, proliferation and immunosuppression efficacy of the clinical treatment using the UCMSCs treated with PG2 has yet to be tested on human beings. Human biological processes may affect the promised effect of PG2 on UCMSCs. Thus, future research has to be done to ensure safety and efficacies for the use of PG2 enhanced UCMSCs. Nevertheless, the studies provided a source for improving UCMSCs proliferation and immunosuppression.
Introduction
Over the last decade, there is an increasing use of Mesenchymal stem cells (MSCs) in clinical application for diseases associated with immune reactions. MSCs are cells that can be isolated from bone marrow and umbilical cords. The collection of MSCs from the umbilical cord is preferred as it is unlike harvesting from bone marrow, it is non-invasive and painless.
Studies have proven that MSCs derived from umbilical cord (UCMSCs) increases more rapidly and better than those derived from bone-marrow (BMMSCs). UCMSCs were also found to have a more effective immunosuppressive response than the BMMSCs which emphasized the importance of developing the biological properties of UCMSC. However, the proliferative potential of MSCs decrease gradually with in-vitro passaging. Clinical efficacy and immunosuppressive capacity of MSCs were impacted due to MSCs senescence that occurs during culture.
A popular traditional Chinese medicine, Astragalus membranaceus was added to determine the enhancement effect on the immunosuppressive and proliferative capacities of MSCs. Astragalus membranaceus is a health-promoting herb that was confirmed to have beneficial biological functions such as vital energy reinforcement . Astragalus membranaceus roots contain Astragalus polysaccharides (APS) was confirmed to have beneficial biological functions such as vital energy reinforcement . Therefore, PG2 reagent that made of APS mixture was added to MSCs to determine the APS effects on MSCs proliferation and its other important biological functions.
Methods
The effects of APS in PG2 reagent on the biological functions of UCMSCs and peripheral blood mononuclear cell (PBMC) were determined. Critical biological functions such as proliferation morphology, differentiation, surface marker expression and immunosuppressive capacities were compared. In order to determine UCMSCs proliferative potential, a proliferative capacity assessment was conducted. Following immunophenotyping test to determine the immunophenotyping profile and immunophenotyping test to determine the immunophenotyping profile. Differential potential was also assessed for osteogenic and adipogenic potential. In addition, PBMC proliferation assay was conducted to measure the influence of PG2 on the suppressive effects of MSCs on PBMC proliferation. Lastly, immunosuppressive capacity was measured by one-way ANOVA and data were analysed using SPSS.
Results and discussion
Differentiation potential, in-vitro morphology and surface marker expression were used as indicator for umbilical cord MSCs identification (UCMSCs). In accordance with the criteria of the International Society for Cellular Therapy, UCMSC appeared as spindle-shaped fibroblastic morphology and underwent induction condition, UCMSCs achieved osteogenic and adipogenic differentiation. Basic properties of UCMSCs were evaluated to determine the influence of PG2. Basic properties consists of morphology, surface marker expression and differentiation potential. The PG2 treatment did not influence the morphology as the spindle-shape was maintained in the PG2-treated UCMSCs (Fig 1).
Figure 1, morphology of control UCMSCs and PG2-treated UCMSCs.
Both UCMSCs treated with or without PG2 has a consistent unique immunophenotypic profile by showing positive for surface markers CD29, CD44, CD73, CD90 and CD105 as well as human leukocyte antigen HLA-A’, HLA-B’ and negative for CD34, CD14 and HLA-DR. By the ALP activity and Oil red O stain conducted under induction conditions, both achieved osteogenic and adipogenic differentiation respectively (Fig 2). Spectrophotometry had further quantified to confirm that there was no alteration in the capacities of UCMSCs for osteogenic and adipogenic differentiation.
Figure 2 Osteogenic (left) & adipogenic (right) differentiation potential.
Significantly higher population doubling was achieved by the UCMSCs that was exposed to the medium containing PG2 and this indicated positive effects on UCMSCs proliferation. Furthermore, the suppressive effects of PG2 treated UCMSCs on PBMC proliferation was increased significantly when phytohemagglutinin (PHA) was stimulated causing a reduction in the proliferation of PBMC. Further assessment was carried out by comparing PG2 treated- UCMSCs and control UCMSCs on CLP- induced septic peritonitis mice. Inflammation-associated cytokine levels were observed to be significantly lower compared to the mice with UCMSCs without PG2 treatment.
In-vitro and in-vivo immunosuppressive capacities as well as proliferation of UCMSCs was enhanced by PG2 reagent with no alteration in their surface marker expression, morphology and differential potential. This is a significant result as PG2 reagent which contains APS is the first plant product that is reported to have an influence on the immunosuppressive and proliferative capacities of the MSC at that point of time when the study was conducted . This could suggest that the APS in PG2 reagent has an useful role on the human MSCs which can be used for future clinical studies.
The main bioactive ingredient in the Astragalus membranaceus is the APG. With the established fact that Astragalus membranaceus is used for vital energy deficiency management, the present study conducted also found that it could reinforces vital energy in the MSCs as well since APG is able to cause an increase in in- vitro and in-vivo immunosuppressive capacities and proliferative. With a better UCMSCs proliferative rate by the higher population doublings caused by PG2 treatment, it would allow the acquisition of cells in a shorter period of time and the collection of younger cells of earlier passages. The study suggested it would be useful for the addition of PG2 into the MSCs culture medium in preparation for clinical use.
MSCs have been investigated for the management of the aberrant immune responses associated clinical diseases due to the interactions between MSCs and a variety of immune cells . An amplified inhibition effects if PBMC proliferation in the PG2 treated UCMSCs that caused a speculation that there could be increased immunosuppressive capacities of UCMSCs due to the addition of PG2. Furthermore, a study of immunosuppressive capacities was conducted on mice that received PG2 treated UCMSCs and mice that only received control UCMSCs. Significantly lower circulating levels of inflammation-associated cytokines were observed in mice that received PG2-treated UCMSCs. Studies successfully demonstrated that there was an increased in-vitro and in-vivo immunosuppressive capacities of UCMSCs which supported the hypothesis made by the authors.
Conclusion
The study has provided experimental results to support the use of Astragalus membranaceus as the first plant product for enhancement of UCMSCs. PG2 component found in APS that is extracted from Astragalus membranaceus could improve the proliferation and in-vitro and in-vivo immunosuppressive capacities of UCMSCs which could provide a better stem cell clinical treatment. Further studies could be done to ensure that the human biological processes does not affect the efficacy of PG2 when used for clinical treatments of aberrant immune responses associated diseases. In addition, comparison of animal products and PG2 efficacy on immunosuppressive and proliferative capacities of the MSCs to determine the suitability of PG2 as a plant product for its purpose.