Elsevier

Cytotherapy

Volume 11, Issue 6, 2009, Pages 688-697
Cytotherapy

Analysis of changes in the viability and gene expression profiles of human mesenchymal stromal cells over time

https://doi.org/10.3109/14653240902974032Get rights and content

Background aims

Because of their ability to differentiate and widespread availability, human mesenchymal stromal cells (hMSC) are often used as a clinical therapeutic tool. However, the factors that determine the quality and viability of hMSC are not well understood.

Methods

We evaluated the viability of hMSC over time using flow cytometry analysis (FACS) and transmission electron microscopy (TEM) to determine if morphologic changes occurred in hMSC. In addition, we conducted gene expression prof ling using an Affymetrix Human Genome U133 Plus 2.0 Array.

Results

FACS analysis revealed that 83% and 76% of the cells were viable in sterilized phosphate-buffered saline (PBS) after 6 h and 12 h, respectively.

TEM data revealed that the total number of cells with healthy chromatins or a few cytosolic vacuoles was significantly reduced over time. We then conducted gene expression prof ling using a microarray, which revealed changes in the expression of 2949 functional genes. Specifcally, among the total of 50 000 gene probes evaluated, the expression levels of apoptosis and stress-related genes were significantly increased over time.

Conclusions

The results of this study suggest that the viability of hMSC decreases after disassociation from the culture dish and time is an essential factor when considering hMSC as a potential source for stem cell-based direct transplantation.

Introduction

Human mesenchymal stromal cells (hMSC) have been investigated for their efficacy as a clinical therapeutic tool in patients with stroke, myocardial infarction, limb ischemia and multiple system atrophy (MAS) [1., 2., 3., 4.] because of their availability and differentiation plasticity. hMSC have been isolated from a variety of tissues, including placenta, adipose tissue and bone marrow [5., 6., 7., 8.]. In addition, it is well known that MSC are multipotent and have the potential to differentiate into osteoblasts, chondroblasts, adipocytes and neurons both in vitro and in vivo [9., 10., 11., 12., 13.], as well as the ability to support hematopoesis [14]. Furthermore, recent studies have shown that hMSC may be useful in tissue engineering [15].

It has been suggested that neurologic disorders can be treated by direct transplantation of stem cells or their derivatives into the adult brain [16]. We have reported previously that intravenous injection of ex vivo -cultured autol-ogous hMSC into patients suffering from ischemic stroke and MAS had the potential to aid in their functional recovery [14]. In addition to being a safe and feasible method of treatment for brain diseases, there are no ethical problems associated with the use of autologous hMSC.

Li et al. 17 reported that approximately 1–5% of transplanted hMSC express proteins that are phenotypic of brain parenchymal cells. Transplanted hMSC often have poor survival rates, which may occur as a result of mechanical trauma, free radicals, deprivation of growth factors and time following cell preparation [18,19]. hMSC have a high potential for cell proliferation, differentiation and adhesion capacity immediately after harvest [20]. Although the viability of hMSC for transplantation has not been investigated thoroughly, it is well known that hMSC should be kept in a fresh condition by minimizing stress and damage prior to transplantation. Therefore, it is important to use hMSC as soon as possible following harvest to improve the chances of successful transplantation.

We conducted this study to analyze the viability and freshness of isolated hMSC that were stored in phosphate-buffered saline (PBS) prior to clinical applications. Although standard culture conditions and techniques have been developed to optimize the growth of hMSC, these conditions are inherently stressful to many other types of stem cells. Therefore, we evaluated changes in the viability and morphology of hMSC over time using flow fluorescence-activated cell sorting (FACS) and transmission electron microscopy (TEM) analysis, respectively. In addition, we utilized complementary (c)DNA microarray analysis to evaluate the effects of storage on the expression of genes by hMSC.

Section snippets

Cell culture

hMSC were obtained from 20-mL aspirates from the iliac crest of normal human donors [1 21]. The procedure used in this study was approved by the Scientif c Ethical Review Board of Ajou University Medical Center (AJIRB-CRO-05–126; Suwon, South Korea). Briefly, human BM-derived MSC were aspirated from the human iliac crest and separated by 70% Percoll-gradient centrifugation. The cells in the low-density fraction were then washed with low-glucose Dulbecco's modified Eagle's medium (DMEM;

Analysis of cell viability by FACS

To determine the level of apoptosis and necrosis in response to incubation in PBS for various lengths of time, we labeled hMSC with Annexin V and PI, respectively, and then evaluated the samples by FACS analysis. Cells incubated in PBS for 3, 6 and 12 h showed a higher degree of apoptosis and necrosis than control cells (Figure 1), although the FACS data did indicate that control cells exhibited a low level of apoptosis. Specifically the viability of the cells at each time-point was as follows:

Discussion

We used FACS and microarray analysis to evaluate the viability and gene expression prof le of hMSC over time. The results of these analyzes revealed that time is a critical factor that may impact the clinical application of hMSC.

FACS analysis revealed that 83% and 76% of cells were viable after 6 and 12 h of incubation, respectively. This suggests that with increasing time in PBS, cells became more sensitive to any treatment or manipulation, such as preparation for FACS. However, when cell

Acknowledgements

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund; KRF-2007–313-H00012) to Gwang Lee, and partly supported by a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean Government (MOST; R01–2007–000–20533–0) to Sangdun Choi.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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