Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine

BACKGROUND Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages.In humans,their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs.Studies suggested that mesenchymal stem cells(MSCs),necessary for repair and regeneration via transplantation,require doses ranging from 10 to 400 million cells.Furthermore,the limited expansion of MSCs restricts their therapeutic application.AIM To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols.METHODS Human umbilical cord(hUC)tissue derived MSCs were obtained and re-cultured.These cultured cells were subjected to the following evaluation pro-cedures:Immunophenotyping,immunocytochemical staining,trilineage differentiation,population doubling time and number,gene expression markers for proliferation,cell cycle progression,senescence-associatedβ-galactosidase assay,human telomerase reverse transcriptase(hTERT)expression,mycoplasma,cytomegalovirus and endotoxin detection.RESULTS Analysis of pluripotent gene markers Oct4,Sox2,and Nanog in recultured hUC-MSC revealed no significant differences.The immunophenotypic markers CD90,CD73,CD105,CD44,vimentin,CD29,Stro-1,and Lin28 were positively expressed by these recultured expanded MSCs,and were found negative for CD34,CD11b,CD19,CD45,and HLA-DR.The recultured hUC-MSC population continued to expand through passage 15.Proliferative gene expression of Pax6,BMP2,and TGFb1 showed no significant variation between recultured hUC-MSC groups.Nevertheless,a significant increase(P<0.001)in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs.Cellular senescence markers(hTERT expression andβ-galactosidase activity)did not show any negative effect on recultured hUC-MSCs.Additionally,quality control assessments consistently confirmed the absence of mycoplasma,cytomegalovirus,and endotoxin contamination.CONCLUSION This study proposes the development of a novel protocol for efficiently expanding stem cell population.This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.

Phase I study on the safety and preliminary efficacy of allogeneic mesenchymal stem cells in hypoxic-ischemic encephalopathy

BACKGROUND Hypoxic-ischemic encephalopathy(HIE)is a leading cause of morbidity and mortality in the adult as well as in the neonate,with limited options for treatment and significant dysfunctionality.AIM To investigate the safety and preliminary efficacy of allogeneic mesenchymal stem cells(MSCs)in HIE patients.METHODS Patients who had HIE for at least 6 mo along with significant dysfunction and disability were included.All patients were given Wharton’s jelly-derived MSCs at 1×106/kg intrathecally,intravenously,and intramuscularly twice a month for two months.The therapeutic effects and prognostic implications of MSCs were evaluated by multiple follow-ups.Functional independence measure(FIM),modified Ashworth,and Karnofsky scales were used to assess any side effects,neurological and cognitive functions,and overall outcomes.RESULTS The 8 subjects included in the study had a mean age of 33.25±10.18 years.Mean HIE exposure and mean post-HIE durations were 45.63±10.18 and 19.67±29.04 mo,respectively.Mean FIM score was 18.38±1.06,mean modified Ashworth score was 43.5±4.63,and mean Karnofsky score was 20.For the first 24 h,5 of the patients experienced a subfebrile state,accompanied by mild headaches due to intrathecally administration and muscle pain because of intramuscularly administration.Neurological and functional examinations,laboratory tests,electroencephalography,and magnetic resonance imaging were performed to assess safety of treatment.Mean FIM score increased by 20.88±3.31 in the first month(P=0.027)and by 31.38±14.69 in 12 mo(P=0.012).The rate of patients with an FIM score of 126 increased from 14.58%to 16.57%in the first month and 24.90%in 12 mo.CONCLUSION Multiple triple-route Wharton’s jelly-derived MSC administrations were found to be safe for HIE patients,indicating neurological and functional improvement.Based on the findings obtained here,further randomized and placebo research could be performed.

Rapid and Cost-Effective Gene Targeting in Rat Embryonic Stem Cells by TALENs

The rat is the preferred animal model in many areas of biomedical research and drug development. Genetic manipulation in rats has lagged behind that in mice due to the lack of efficient gene targeting tools. Previously, we generated a knockout rat via conventional homologous recombination in rat embryonic stern （ES） cells. Here, we show that efficient gene targeting in rat ES cells can be achieved quickly through transcription activator-like effector nuclease （TALEN）-mediated DNA double-strand breaks. Using the Golden Gate cloning technique, we constructed a pair of TALEN targeting vectors for the gene of interest in 5 days. After gene transfection, the targeted rat ES cell colonies were isolated, screened, and confirmed by PCR without the need of drug selection. Our results suggest that TALEN-mediated gene targeting is a superior means of establishing genetically modified rat ES cell lines with high efficiency and short turnaround time.

Induced Neural Stem Cells Generated from Rat Fibroblasts

The generation of induced tissue-specific stem cells has been hampered by the lack of well-established methods for the maintenance of pure tissue-specific stem cells like the ones we have for embryonic stem （ES） cell cultures. Using a cocktail of cytokines and small molecules, we dem- onstrate that primitive neural stem （NS） cells derived from mouse ES cells and rat embryos can be maintained. Furthermore, using the same set of cytokines and small molecules, we show that induced NS （iNS） cells can be generated from rat fibroblasts by forced expression of the transcrip- tional factors Oct4, Sox2 and c-Myc. The generation and long-term maintenance of iNS cells could have wide and momentous implications.

Neuroendocrine differentiation of prostate cancer

Benign prostate and prostatic adeno- carcinoma contain rare quiescentneuroendocrine cells while small cell neu- roendocrine carcinoma （SCNC）, a variant form of prostate cancer, contains highly proliferative neuroendocrine tumor cells. We provide evidence that IL-8-CXCR2- P53 pathway keeps the NE cells in a quiescent state normally. P53 mutation inactivates this pathway, resulting in hyper-proliferation and aggressive beha- vior of the NE cells in SCNC. Therefore, we have identified potential cells of origin and a molecular target for prostatic SCNC that are different from those of adenocarci- noma, which explains SCNC＇s distinct bio- logy and the clinical observation that it does not respond to hormonal therapy.

Metabolism of pluripotent stem cells

BACKGROUND： Recently, growing attention has been directed toward stem cell metabolism, with the key observation that metabolism not only fuels the proper functioning of stem cells but also regulates the fate of these cells. There seems to be a clear link between the self-renewal ofpluripotent stem cells （PSCs）, in which cells proliferate indefinitely without differentiation, and the activity of specific metabolic pathways. The unique metabolism in PSCs plays an important role in maintaining pluripotency by regulating signaling pathways and resetting the epigenome. OBJECTIVE： To review the most recent publications concerning the metabolism of pluripotent stem cells and the role of metabolism in PSC self-renewal and differentiation. METHODS： A systematic literature search related to the metabolism of PSCs was conducted in databases including Medline, Embase, and Web of Science. The search was performed without language restrictions on all papers published before May 2016. The following keywords were used： ＂metabolism＂ combined with either ＂embryonic stem cell＂ or ＂epiblast stem cell.＂ RESULTS： Hundreds of papers focusing specifically on the metabolism of pluripotent stem cells were uncovered and summarized. CONCLUSION： Identifying the specific metabolic pathways involved in pluripotency maintenance is crucial for progress in the field of developmental biology and regenerative medicine. Additionally, better understanding of the metabolism in PSCs will facilitate the derivation and maintenance of authentic PSCs from species other than mouse, rat, and human.

Long-term, in vivo therapeutic effects of a single dose of miR-145 micelles for atherosclerosis

Atherosclerosis is a chronic inflammatory disease that is characterized by the build-up of lipid-rich plaques in the arterial walls. The standard treatment for patients with atherosclerosis is statin therapy aimed to lower serum lipid levels. Despite its widespread use, many patients taking statins continue to experience acute events. Thus, to develop improved and alternative therapies, we previously reported on microRNA-145 (miR-145 micelles) and its ability to inhibit atherosclerosis by targeting vascular smooth muscle cells (VSMCs). Importantly, one dose of miR-145 micelles significantly abrogated disease progression when evaluated two weeks post-administration. Thus, in this study, to evaluate how long the sustained effects of miR-145 micelles can be maintained and towards identifying a dosing regimen that is practical for patients with chronic disease, the therapeutic effects of a single dose of miR-145 micelles were evaluated for up to two months in vivo. After one and two months post-treatment, miR-145 micelles were found to reduce plaque size and overall lesion area compared to all other controls including statins without causing adverse effects. Furthermore, a single dose of miR-145 micelle treatment inhibited VSMC transdifferentiation into pathogenic macrophage-like and osteogenic cells in plaques. Together, our data shows the long-term efficacy and sustained effects of miR-145 micelles that is amenable using a dosing frequency relevant to chronic disease patients.