Preclinical Safety Study Of Umbilical Cord Mesenchymal Stem Cells

Mesenchymal stem cells, pocessed multipotency and immunomodulatory, were used in regenerative medicine and the treatment of autoimmune diseases in many clinical trials. Different from Hematopoietic Stem Cells (HSCs), MSCs could be expanded in vitro. The incidence of MSCs in tissue is extremely low, but MSCs could propagate in vitro to achieve the quantity for clinical requirement. No serious MSCs-related complication was observed in clinical trials. However, we still know litter about MSCs, especially about MSCs which were expanded in vitro for a long term. More toxity studies were needed to evaluate the safety and efficiency of MSCs for clinical application.The current studies investigated the effect of long-term culture in vitro on the biological characteristics of hUC-MSCs, including multipotency, spontaneous malignant transformation, transcriptome and genome stability. The data of this research were useful for evaluating the safety of hUC-MSCs for clinical application.Part I:Long-Term Expansion and Biological Characteristics Alternations of hUC-MSCsTo determine if in vitro expansion will influence the genetic stability and other biological characteristics of hUC-MSCs, we maintained hUC-MSCs in long-term culture. After variable in vitro life span, all of hUC-MSCs involved in this study developed a senescence phase. None of our hUC-MSCs samples underwent spontaneous malignant transformation after long-term in vitro culture. The expression of hTERT was very low in hUC-MSCs at early-passage, and decreased in the process of in vitro culture. Unequal genetic alternations were identified among different hUC-MSCs lines during the process of long-term culture. Two hUC-MSCs samples showed no DNA copy number variations (CNVs) at late-passage, seven (78%) had one or more CNVs at the30th passage compared to the3rd passage and among which one hUC-MSCs appeared a trisomic of chromosome10at the30th passage. No matter whether gained CNVs during long-term culture, hUC-MSCs at the30th passage could not form tumor in immunodeficiency mice, even with the support of Matrigel. Transcriptome analysis based on mRNA deep sequencing revealed that hUC-MSCs with different genetic stability processed different trascriptome profiles. During long-term in vitro culture, pathways related cell cycle control and DNA damage response were down-regulated in the hUC-MSCs samples which got trisomic of chromosome10at the30th passage. However, these same pathways were up-regualted in hUC-MSCs samples without CNVs at the30th passage.These results indicate that hUC-MSCs can be large-scale expanded but most of the cultured hUC-MSCs will develop some genetic alternations during in vitro culture. Although these cells with genetic instability will not certainly undergo tumor transformation, periodic genomic monitoring was recommended before they are used for clinical trials.Part II:Long-term Culture of Human Umbilical Cord Mesenchymal Stem Cells in Chemical Defined Serum Free MediumClassic medium for MSCs, even for clinical using, contained fetal calf serum. Due to the risk of contamination of infectious pathogens, serum-containing medium (SCM) are a major obstacle for MSCs-related therapies. Some studies demonstrated that MSCs could be expanded in serum free medium (SFM), but whether SFM would change the biological characteristics and safety issues of MSCs has not been well answered. Therefore, we cultured human umbilical cord mesenchymal stem cells (hUC-MSCs) in a chemical defined serum free medium. Growth rate, in vitro life span, multipotency, surface antigen expression, telomerase, transcriptome and genome stability of SFM derived hUC-MSCs were investigated. SFM-expanded hUC-MSCs maintained multipotency and the profile of cell surface antigen expression which were used to define human MSCs. hUC-MSCs propagated more slowly and senesce earlier in SFM compared with SCM. SFM-expanded hUC-MSCs were different from SCM-expanded hUC-MSCs in transcriptome profile. The most changed genes in SCM-expanded hUC-MSCs were enriched in the pathway of cell cycle, regulation of actin cytoskeleton, MAPK signaling. Both SFM-and SCM-expanded hUC-MSCs gained copy number variation (CNV) in long-term in vitro culture.Our data suggested that hUC-MCSs could be safely expanded in chemical defined serum free medium to obtain enough cells without any indication of malignant transformation, thus rendering SFM-derived hUC-MSCs were suitable for clinical applications.Part Ⅲ:A Toxicity Study of Multiple-Administration Human Umbilical Cord Mesenchymal Stem Cells in Cynomolgus MonkeysTherapies based on stem cells have shown very attractive potential in many clinical studies. However, the data about the safety of stem cells application remains insufficient. The present study was designed to evaluate the overall toxicology of human umbilical cord mesenchymal stem cells (hUC-MSCs) in cynomolgus monkeys with repeated administrations.hUC-MSCs were administered by intravenous injection once every2weeks for6 weeks (4times in total). The dose levels employed in this study were2×106,1×107cells/kg body weight. Toxicity was evaluated by clinical observations (body weight, body temperature, and ophthalmology exams), pathology (blood cell counts, clinical biochemistry, urinalysis, and bone marrow smears), immunologic consequences (lymphoproliferation assay, the secretion of interferon-y and interleukin-4, the percentage of CD3+, CD4+, CD8+T cells, and the ratio of CD4+and CD8+T cells) and anatomic pathology. Pharmacodynamics in blood and distribution of hUC-MSCs in the tissues of cynomolgus monkeys were measured by real-time PCR.All animals survived until scheduled euthanasia. No stem cells transplantation-related toxicity was found in this study. hUC-MSCs could be found in the blood of cynomolgus monkeys beyond8h. The findings of this6-week toxicity study showed that the transplantation of hUC-MSCs did not affect the general health of cynomolgus monkeys.