Xiuying Li

Comprehensive characterization of four different populations of human mesenchymal stem cells as regards their immune properties, proliferation and differentiation

In the present study, we compared mesenchymal stem cells (MSCs) derived from 4 different sources, human bone marrow (BM), adipose tissue (AT), umbilical cord Wharton’s Jelly (WJ) and the placenta (PL), in order to determine which population of MSCs displayed the most prominent immunosuppressive effects on phytohemagglutinin-induced T cell proliferation, and which one had the highest proliferative and differentiation potential. MSC and T lymphocyte co-culture (mixed culture) was used to determine whether the MSCs inhibit T cell proliferation, as well as which population of MSCs has the strongest inhibitory ability. The expression of immune-related genes was analyzed by RT-PCR and RT-qPCR. The proliferation and differentiation potential of the MSCs were determined using standard methods. Following MSC and T cell co-culture, mitogen-induced T cell proliferation was effectively suppressed by all 4 populations of MSCs. This occurred through soluble factors rather than direct contact inhibition. Among the 4 populations of MSCs, the WJ-MSC has the strongest suppression effects. On immune related genes, WJ-MSC has the weakest expression of MHC II genes, TLR4, TLR3, JAG1, NOTCH2 and NOTCH3. To compare the proliferation potential, WJ-MSCs showed the most rapid growth rate followed by the AT-, PL- and BM-MSCs. As regards differentiation potential, the WJ-MSCs had the strongest osteogenetic ability followed by PL, AT and BM-MSC. AT-MSC has the strongest adipogenetic ability followed by the WJ-, BM- and PL-MSCs. These data indicated that the WJ-MSCs had the strongest immunomodulatory and immunosuppressive potential. In light of these observations, we suggest that WJ-MSCs are the most attractive cell population for use in immune cellular therapy when immunosuppressive action is required.

Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR

Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases.

Identification of optimal reference genes for quantitative PCR studies on human mesenchymal stem cells

Quantitative polymerase chain reaction (qPCR) analysis is a commonly used method for the study of mRNA expression throughout the field of mesenchymal stem cell (MSC) research. This technology is simple and sensitive; however the results may vary significantly due to the use of various reference genes (RGs) as normalizers. Therefore, the reliable use of RGs is vital for obtaining accurate results. The present study focuses on ten putative RGs for the normalization of qPCR data between human bone marrow-derived MSCs (BM-MSCs) and fetal tissue-derived MSCs (FT-MSCs). The total RNA from these two types of MSC was isolated using TRIzol reagent. cDNA was generated from the RNA via reverse transcription and subsequently analyzed by qPCR using ten common RGs as normalizers. These RGs included 18S, ACTB, B2M, HPRT1, GAPDH, TBP, PPIA, RPLP0, PGK1 and RPL13A. GeNorm, NormFinder and BestKeeper software were used to analyze the qPCR results by evaluating the expression stabilities of the ten candidate RGs in BM-MSCs and FT-MSCs. Consequently, several of the commonly used RGs, including 18S, ACTB and TBP, were demonstrated to be unsuitable for normalization in these two MSCs, whereas RPL13A, B2M and PPIA were the most stable RGs and were therefore reliable for use in qPCR studies. Combining multiple RGs had no contribution towards increasing their stabilities. In conclusion, the present study revealed that RPL13A, B2M and PPIA were the optimal RGs for qPCR studies comparing BM-MSCs and FT-MSCs.

Umbilical Cord Tissue-Derived Mesenchymal Stem Cells Induce T Lymphocyte Apoptosis and Cell Cycle Arrest by Expression of Indoleamine 2, 3-Dioxygenase.

It has been reported that human mesenchymal stem cells are able to inhibit T lymphocyte activation; however, the discrepancy among different sources of MSCs is not well documented. In this study, we have compared the MSCs from bone marrow (BM), adipose tissue (AT), placenta (PL), and umbilical cord (UC) to determine which one displayed the most efficient immunosuppressive effects on phytohemagglutinin-induced T cell proliferation. Among them we found that hUC-MSC has the strongest effects on inhibiting T cell proliferation and is chosen to do the further study. We observed that T lymphocyte spontaneously released abundant IFN-γ. And IFN-γ secreted by T lymphocyte could induce the expression of indoleamine 2, 3-dioxygenase (IDO) in hUC-MSCs. IDO was previously reported to induce T lymphocyte apoptosis and cell cycle arrest in S phase. When cocultured with hUC-MSCs, T lymphocyte expression of caspase 3 was significantly increased, while Bcl2 and CDK4 mRNA expression decreased dramatically. Addition of 1-methyl tryptophan (1-MT), an IDO inhibitor, restored T lymphocyte proliferation, reduced apoptosis, and induced resumption of the cell cycle. In addition, the changes in caspase 3, CDK4, and Bcl2 expression were reversed by 1-MT. These findings demonstrate that hUC-MSCs induce T lymphocyte apoptosis and cell cycle arrest by expressing abundant IDO and provide an explanation for some of the immunomodulatory effects of MSCs.

Identification of suitable reference genes for gene expression studies using quantitative polymerase chain reaction in lung cancer in vitro

The present study aimed to examine 10 housekeeping genes (HKGs), including 18s ribosomal RNA (18S), glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH), ribosomal protein large P0 (RPLP0), β‑actin (ACTB), peptidylprolyl isomerase A (PPIA), phosphoglycerate kinase‑1 (PGK1), β‑2‑microglobulin (B2M), ribosomal protein LI3a (RPL13A), hypoxanthine phosphoribosyl transferase‑1 (HPRT1) and TATA box binding protein (TBP) in order to identify the most stable and suitable reference genes for use in expression studies in non‑small cell lung cancer. The mRNA expression encoding the panel of the 10 HKGs was determined using reverse transcription‑quantitative PCR (RT‑qPCR) in human lung cancer cell lines. Three software programs, BestKeeper, NormFinder and geNorm, were used to ascertain the most suitable reference genes to normalize the RNA input. The present study examined three lung cancer cell lines (A549, NCI‑H446 and NCI‑H460). The analysis of the experimental data using BestKeeper software revealed that all 10 HKGs were stable, with GADPH, followed by 18S being the most stable genes and PPIA and HPRT1 being the least stable genes. The NormFinder software results demonstrated that PPIA followed by ACTB were the most stable and B2M and RPLP0 were the least stable. The geNorm software results revealed that ACTB and PGK1, followed by PPIA were the most stable genes and B2M and RPLP0 were identified as the least stable genes. Due to discrepancies in the ranking orders of the reference genes obtained by different analyzing software programs, it was not possible to determine a single universal reference gene. The suitability of selected reference genes requires unconditional validation prior to each study. Based on the three analyzing programs, ACTB, PPIA and PGK1 were the most stable reference genes in lung cancer cell lines.

Evaluation and validation of the suitable control genes for quantitative PCR studies in plasma DNA for non‑invasive prenatal diagnosis

Quantitative polymerase chain reaction (qPCR) is widely used in quantitation of plasma DNA for non‑invasive prenatal diagnosis (NIPD). Control genes are indispensable as standard normalizers in qPCR analysis, and there is increasing evidence indicating that the content levels of commonly used control genes vary significantly in different independent experiments. The commonly used control genes for DNA quantitation using qPCR in plasma DNA analysis are frequently chosen without any preliminary evaluation of their suitability. The present study aimed to examine a panel of six common control genes (HBB, TERT, GAPDH, ALB, ACTB and TRG) in order to evaluate and validate the most reliable control genes for qPCR studies in the quantitation of plasma DNA from pregnant and non‑pregnant females for NIPD. Plasma DNA was extracted from the peripheral blood of 18 pregnant females and 18 non‑pregnant females by the QIAamp DNA mini kit. qPCR followed by geNorm, NormFinder and BestKeeper based analysis was conducted to evaluate the DNA content stabilities of the six candidate control genes. DSCR3 was used to validate the result. The study recommended TERT and the combination of ACTB and TERT as the optimal control genes for qPCR studies on pregnant/non‑pregnant plasma DNA quantitation. Thus, the study reveals that the DNA content stability of widely used control genes varies significantly in pregnant and non‑pregnant plasma DNA.

Heme oxygenase-1 silencing increases the sensitivity of human osteosarcoma MG63 cells to arsenic trioxide

Arsenic trioxide (ATO) has been successfully used to treat leukemia and some solid malignant tumors. Our previous study regarding the effects of ATO on mesenchymal-derived human osteosarcoma MG63 cells showed that heme oxygenase-1 (HO-1) was strongly induced upon treatment with ATO. The present study sought to investigate the effect of silencing HO-1 on the sensitivity of osteosarcoma cells to ATO to determine the potential for therapeutic applications. Small hairpin RNA (shRNA)-mediated interference was used to silence HO-1 in MG63 cells. Viability, apoptosis, and intracellular reactive oxygen species (ROS) of the cells were assessed to evaluate the sensitivity of the cells to ATO as well as the potential mechanisms responsible. shRNA-mediated interference prevented the induction of HO-1, increased cell death, and increased intracellular ROS levels in MG63 cells upon treatment with ATO. Silencing HO-1 increased the susceptibility of MG63 cells to the chemotherapeutic drug ATO by enhancing intracellular accumulation of ROS. Our results suggest that the inhibition of HO-1 could improve the outcome of osteosarcoma treated with ATO.

Evaluation of suitable control genes for quantitative polymerase chain reaction analysis of maternal plasma cell-free DNA

The content stability of commonly used control genes is considered to vary significantly in different independent experimental systems, either in the expression of RNA expression or in the level of DNA content. The present study aimed to examine a panel of six common control genes, including β‑globin (HBB), telomerase (TERT), glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH), albumin (ALB), β‑actin (ACTB) and T cell receptor γ (TRG), in order to evaluate and validate the most reliable control genes for quantitative polymerase chain reaction (qPCR) in investigations for the analysis of fetal‑derived DNA and maternal‑derived DNA in maternal plasma to enable non‑invasive prenatal assessment. Plasma DNA was extracted from the peripheral blood of 20 pregnant femals (gestational age, 18.67 ± 0.58 weeks) using a QIAamp DNA mini kit. Electrophoresis was performed to separate the fetal‑derived DNA and the maternal‑derived DNA at the 300bp position. qPCR was then performed, followed by geNorm‑, NormFinder‑ and BestKeeper‑based analyses to evaluated the content stabilities of the six candidate control genes in the fetal‑derived DNA and maternal‑derived DNA. The subsequent analysis of the experimental data revealed that HBB was expressed in the maternal‑ and fetal‑derived DNA together and in the maternal‑derived DNA alone. In addition, GAPDH in the fetal‑derived DNA enabled efficient normalization for qPCR investigations in the maternal plasma DNA.

Evaluation of eight reference genes for quantitative polymerase chain reaction analysis in human T lymphocytes co‑cultured with mesenchymal stem cells

Accurate gene expression analysis relies on the selection of a stable reference gene, as unstable reference genes can alter experimental results and conclusions. It is widely‑accepted that reference genes exhibit different expression levels in different types of tissues and cells. Therefore, it is essential to screen for stably‑expressed reference genes in the cells and tissues used for experimental analysis prior to performing reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). In the present study, eight reference genes were screened for their suitability for RT‑qPCR in five T lymphocytes co‑cultured with mesenchymal stem cells from different sources. Using NormFinder, geNorm, and BestKeeper algorithms consistently demonstrated that RPL13A and B2M were the optimal reference genes for the normalization of RT‑qPCR data obtained from T lymphocytes, whereas glyceraldehyde 3‑phosphate dehydrogenase was not a suitable reference gene due to its extensive variability in expression. These findings highlight the importance of evaluating reference genes for RT‑qPCR.