经血源子宫内膜干细胞培养、鉴定及体外分化潜能的研究

现阶段干细胞的来源常具有侵入性,该文旨在研究新来源于经血的经血源子宫内膜干细胞（menstrual blood-derived mesenchymal stem cells,MenSCs）的基本生物学特性及分化潜能。采用密度梯度法从女性经血中分离MenSCs,测定MenSCs群体倍增时间,流式细胞仪鉴定细胞表面抗原,免疫荧光法检测MenSCs nestin阳性表达情况,体外验证其成骨成脂分化潜能。结果表明,MenSCs具有典型的梭状结构,细胞倍增时间为32.2 h,均一地高表达CD29、CD90及CD105,不表达CD14、CD45、HLA-DR。免疫荧光表明,MenSCs为nestin阳性。MenSCs成脂诱导后,油红O染色为阳性。成骨诱导前期诱导组细胞胶原表达量升高,诱导两周后MenSCs形成钙结节,诱导组细胞ALP（alkaline phosphatase）活性连续3周呈上升趋势。以上证明,MenSCs具有来源广泛的优势,具有较高的增殖能力、较低免疫原性、nestin阳性及多向分化潜能等特性,可成为干细胞治疗的理想种子细胞。     

What is the future for cord blood stem cells?

Stem and progenitor cells are present in cord blood at a high frequency making these cells a major target population for experimental and clinical studies. Over the past decade there has been considerable developments in cord blood research and transplantation but despite the rapid progress many problems remain. The initial hope that cord blood would be an alternative source of haemopoietic cells for transplantation has been tempered by the fact that there are insufficient cells in most cord blood collections to engraft an adult of average weight. In attempts to increase the cell number, a plethora of techniques for ex-vivo expansion have been developed.These techniques have also proved useful for gene therapy. As cord blood cells possess unique properties this allows them to be utilised as suitable vehicles for gene therapy and long-term engraftment of transduced cells has been achieved. Current work examining the nature of the stem cells present in this haematological source indicates that cord blood contains not only haemopoietic stem cells but also primitive non-haemopoietic cells with high proliferative and developmental potential. As attention focuses on stem cell biology and the controversies surrounding the potential use of embryonic stem cells in treatment of disease, the properties of stem cells from other sources including cord blood are being re-appraised. The purpose of this article is to review some of the current areas of work and highlight biological problems associated with the use of cord blood cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative capability of menstrual blood versus bone marrow derived stem cells in neural differentiation

In order to characterize the potency of menstrual blood stem cells (MenSCs) for future cell therapy of neurological disorders instead of bone marrow stem cells (BMSCs) as a well-known and conventional source of adult stem cells, we examined the in vitro differentiation potential of these stem cells into neural-like cells. The differentiation potential of MenSCs to neural cells in comparison with BMSCs was assessed under two step neural differentiation including conversion to neurosphere-like cells and final differentiation. The expression levels of Nestin, Microtubule-associated protein 2, gamma-aminobutyric acid type B receptor subunit 1 and 2, and Tubulin, beta 3 class III mRNA and/or protein were up-regulated during development of MenSCs into neurosphere-like cells (NSCs) and neural-like cells. The up-regulation level of these markers in differentiated neural-like cells from MenSCs was comparable with differentiated cells from BMSCs. Moreover, both differentiated MenSCs and BMSCs expressed high levels of potassium, calcium and sodium channel genes developing functional channels with electrophysiological recording. For the first time, we demonstrated that MenSCs are a unique cell population with differentiation ability into neural-like cells comparable to BMSCs. In addition, we have introduced an approach to generate NSCs from MenSCs and BMSCs and their further differentiation into neural-like cells in vitro. Our results hold a promise to future stem cell therapy of neurological disorders using NSCs derived from menstrual blood, an accessible source in every woman.     

Biological characteristics of human menstrual blood‐derived endometrial stem cells

Successful isolation of human endometrial stem cells from menstrual blood, namely menstrual blood‐derived endometrial stem cells (MenSCs), has provided enticing alternative seed cells for stem cell‐based therapy. MenSCs are enriched in the self‐regenerative tissue, endometrium, which shed along the periodic menstrual blood and thus their acquisition involves no physical invasiveness. However, the impact of the storage duration of menstrual blood prior to stem cell isolation, the age of the donor, the number of passages on the self‐renewing of MenSCs, the paracrine production of biological factors in MenSCs and expression of adhesion molecules on MenSCs remain elusive. In this study, we confirmed that MenSCs reside in shedding endometrium, and documented that up to 3 days of storage at 4°C has little impact on MenSCs, while the age of the donor and the number of passages are negatively associated with proliferation capacity of MenSCs. Moreover, we found that MenSCs were actually immune‐privileged and projected no risk of tumour formation. Also, we documented a lung‐ and liver‐dominated, spleen‐ and kidney‐involved organic distribution profile of MenSC 3 days after intravenous transfer into mice. At last, we suggested that MenSCs may have potentially therapeutic effects on diseases through paracrine effect and immunomodulation.     

Production of functional dendritic cells from menstrual blood��a new dendritic cell source for immune therapy

Dendritic cells (DCs) are the most professional antigen-presenting cells of the mammalian immune system. They are able to phagocytize, process antigen materials, and then present them to the surface of other cells including T lymphocytes in the immune system. These capabilities make DC therapy become a novel and promising immune-therapeutic approach for cancer treatment as well as for cancer vaccination. Many trials of DC therapy to treat cancers have been performed and have shown their application value. They involve harvesting monocytes or hematopoietic stem cells from a patient and processing them in the laboratory to produce DCs and then reintroduced into a patient in order to activate the immune system. DCs were successfully produced from peripheral, umbilical cord blood-derived monocytes or hematopoietic stem cells. In this research, we produced DCs from human menstrual blood-derived monocytes. Briefly, monocytes were isolated by FACS based on FSC vs. SSC plot from lysed menstrual blood. Obtained monocytes were induced into DCs by a two-step protocol. In the first step, monocytes were incubated in RPMI medium supplemented with 2% FBS, GM-CSF, and IL-4, followed by incubation in RPMI medium supplemented with α-TNF in the second step. Our data showed that induced monocytes had typical morphology of DCs, expressed HLA-DR, HLA-ABC, CD80 and CD86 markers, exhibited uptake of dextran-FITC, stimulated allogenic T cell proliferation, and released IL-12. These results demonstrated that menstrual blood can not only be a source of stromal stem cell but also DCs, which are a potential candidate for immune therapy.     

Nanomaterials modulate stem cell differentiation: biological interaction and underlying mechanisms

Stem cells are unspecialized cells that have the potential for self-renewal and differentiation into more specialized cell types. The chemical and physical properties of surrounding microenvironment contribute to the growth and differentiation of stem cells and consequently play crucial roles in the regulation of stem cells’ fate. Nanomaterials hold great promise in biological and biomedical fields owing to their unique properties, such as controllable particle size, facile synthesis, large surface-to-volume ratio, tunable surface chemistry, and biocompatibility. Over the recent years, accumulating evidence has shown that nanomaterials can facilitate stem cell proliferation and differentiation, and great effort is undertaken to explore their possible modulating manners and mechanisms on stem cell differentiation. In present review, we summarize recent progress in the regulating potential of various nanomaterials on stem cell differentiation and discuss the possible cell uptake, biological interaction and underlying mechanisms.     

Isolation, characterization, and mesodermic differentiation of stem cells from adipose tissue of camel (Camelus dromedarius)

Adipose-derived stem cells are an attractive alternative as a source of stem cells that can easily be extracted from adipose tissue. Isolation, characterization, and multi-lineage differentiation of adipose-derived stem cells have been described for human and a number of other species. Here we aimed to isolate and characterize camel adipose-derived stromal cell frequency and growth characteristics and assess their adipogenic, osteogenic, and chondrogenic differentiation potential. Samples were obtained from five adult dromedary camels. Fat from abdominal deposits were obtained from each camel and adipose-derived stem cells were isolated by enzymatic digestion as previously reported elsewhere for adipose tissue. Cultures were kept until confluency and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteogenic, and chondrogenic potential. The morphology of resultant camel adipose-derived stem cells appeared to be spindle-shaped fibroblastic morphology, and these cells retained their biological properties during in vitro expansion with no sign of abnormality in karyotype. Under inductive conditions, primary adipose-derived stem cells maintained their lineage differentiation potential into adipogenic, osteogenic, and chondrogenic lineages during subsequent passages. Our observation showed that like human lipoaspirate, camel adipose tissue also contain multi-potent cells and may represent an important stem cell source both for veterinary cell therapy and preclinical studies as well.     

Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants

Purging of tumor cells and selection of stem cells are key technologies for enabling stem cell transplantation and stem cell gene therapy. Here we report a strategy for cell selection based on physical properties of the cells. Exposing cells to an external pulsed electric field (PEF) increases the natural potential difference across the cell membrane until a critical threshold is reached and pore formation occurs, resulting in fatal perturbation of cell physiology. Attaining this threshold is a function of the applied field intensity and cell size, with larger cells porated at lower field intensities than smaller cells. Since hematopoietic stem cells are smaller than other hematopoietic cells and tumor cells, we found that exposure of peripheral blood mononuclear cells (PBMCs) to PEFs caused stepwise elimination of monocytes without affecting the function of smaller lymphocyte populations. Mobilized peripheral blood exposed to PEFs was enriched for CD34+/CD38- cells and stem cell function was preserved. Furthermore, PEF treatment was able to selectively purge blood preparations of tumor cells and eradicate transplantable tumor.     

Carbon Quantum Dots as Multi-Purpose Nanomaterial in Stem Cell Therapy

During stem cell therapy, some issues, such as obscure fate of stem cells or their low survival rate in the body, should be addressed to boost their therapeutic efficiency. Nanotechnology offers a suitable solution to combat such limitations. Carbon quantum dots (CQDs) are carbon-based nanomaterials and may be used as multi-purpose compounds in stem cell therapy. CQDs are excellent choices for stem cell labeling thanks to their special features such as optical properties and good biocompatibility. Besides, they can modulate the biological function of stem cells, such as their proliferation, homing ability, and differentiation properties. Considering the charismatic feature of CQDs and their broad unique effect on stem cells, the current review aims to summarize the most advancements in this field. Hence, we first focused on CQDs synthesis and their applications. In the next section, the stem cell categories will be discussed, and the final part is dedicated to the recent research evaluating the impact of CQDs on stem cell therapy.     

Mesenchymal stem cells as carrier of the therapeutic agent in the gene therapy of blood disorders

Nonhematopoietic stem cells as a delivery platform of therapeutic useful genes have attracted widespread attention in recent years, owing to gained a long lifespan, easy separation, high proliferation, and high transfection capacity. Mesenchymal stem/stromal cells (MSCs) are the choice of the cells for gene and cell therapy due to high self-renewal capacity, high migration rate to the site of the tumor, and with immune suppressive and anti-inflammatory properties. Hence, it has a high potential of safety genetic modification of MSCs for antitumor gene expression and has paved the way for the clinical application of these cells to target the therapy of cancers and other diseases. The aim of gene therapy is targeted treatment of cancers and diseases through recovery, change, or enhancement cell performance to the sustained secretion of useful therapeutic proteins and induction expression of the functional gene in intended tissue. Recent developments in the vectors designing leading to the increase and durability of expression and improvement of the safety of the vectors that overcome a lot of problems, such as durability of expression and the host immune response. Nowadays, gene therapy approach is used by MSCs as a delivery vehicle in the preclinical and the clinical trials for the secretion of erythropoietin, recombinant antibodies, coagulation factors, cytokines, as well as angiogenic inhibitors in many blood disorders like anemia, hemophilia, and malignancies. In this study, we critically discuss the status of gene therapy by MSCs as a delivery vehicle for the treatment of blood disorders. Finally, the results of clinical trial studies are assessed, highlighting promising advantages of this emerging technology in the clinical setting.     

Adipose-derived stem cells decreased microglia activation and protected dopaminergic loss in rat lipopolysaccharide model

Adult stem cell therapy is being used extensively to rejuvenate damaged tissue. One important tissue source to obtain these cells is adipose, which contains cells called adipose-derived stem cells (ADSCs). These cells have a great therapeutic potential not only for their multipotent properties as well as for immunomodulatory effects on the immune system. Parkinson's disease is characterized as neurodegenerative disorder which etiology is undoubtedly related to neuroinflammation process. The properties of ADSCs can be used as a new tool in stem cells therapy to treat neurodegenerative disorders. However, their efficacies are still controversial. Some authors have reported neuroprotection effects, while others did not find differences or stem cells increased the damage. Our previous study showed that ADSCs can survive long time after transplantation, suggesting us some biological effects could need more time to be repaired. In this study, we assessed the neuroprotection 6 months after transplantation. Our results suggest ADSCs can protect the dopaminergic loss after lipopolysaccharide (LPS) injection both reducing the microglia activation and differentiating into dopaminergic cells.     

Heterogeneous phenotype of human glioblastoma: In vitro study

Glioblastomas (GBMs) are the most lethal primary brain tumours. Increasing evidence shows that brain tumours contain the population of stem cells, so‐called cancer stem cells (CSCs). Stem cell marker CD133 was reported to identify CSC population in GBM. Further studies have indicated that CD133 negative cells exhibiting similar properties and are able to initiate the tumour, self‐renew and undergo multilineage differentiation. GBM is a highly heterogeneous tumour and may contain different stem cell populations with different functional properties. We characterized five GBM cell lines, established from surgical samples, according to the marker expression, proliferation and differentiation potential. CD133 positive cell lines showed increased proliferation rate in neurosphere condition and marked differentiation potential towards neuronal lineages. Whereas two cell lines low‐expressing CD133 marker showed mesenchymal properties in vitro, that is high proliferation rate in serum condition and differentiation in mesenchymal cell types. Further, we compared therapy resistance capacity of GBM cell lines treated with hydroxyurea. Our results suggest that CSC concept is more complex than it was believed before, and CD133 could not define entire stem cell population within GBM. At least two different subtypes of GBM CSCs exist, which may have different biological characteristics and imply different therapeutic strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

Targeting ROR1 inhibits the self‐renewal and invasive ability of glioblastoma stem cells

Glioblastoma is the most malignant of brain tumours and is difficult to cure because of interruption of drug delivery by the blood–brain barrier system, its high metastatic capacity and the existence of cancer stem cells (CSCs). Although CSCs are present as a small population in malignant tumours, CSCs have been studied as they are responsible for causing recurrence, metastasis and resistance to chemotherapy and radiotherapy for cancer. CSCs have self‐renewal characteristics like normal stem cells. The aim of this study was to investigate whether receptor tyrosine kinase‐like orphan receptor 1 (ROR1) is involved in stem cell maintenance and malignant properties in human glioblastoma. Knockdown of ROR1 caused reduction of stemness and sphere formation capacity. Moreover, down‐regulation of ROR1 suppressed the expression of epithelial‐mesenchymal transition‐related genes and the tumour migratory and invasive abilities. The results of this study indicate that targeting ROR1 can induce differentiation of CSCs and inhibit metastasis in glioblastoma. In addition, ROR1 may be used as a potential marker for glioblastoma stem cells as well as a potential target for glioblastoma stem cell therapy. Copyright © 2016 John Wiley & Sons, Ltd.     

Placental-derived stem cells: Culture,differentiation and challenges

Stem cell therapy is a promising approach to clinical healing in several diseases. A great variety of tissues (bone marrow, adipose tissue, and placenta) are potentially sources of stem cells. Placenta-derived stem cells (p-SCs) are in between embryonic and mesenchymal stem cells, sharing characteristics with both, such as non-carcinogenic status and property to differentiate in all embryonic germ layers. Moreover, their use is not ethically restricted as fetal membranes are considered medical waste after birth. In this context, the present review will be focused on the biological properties, culture and potential cell therapy uses of placental-derived stem cells. Immunophenotype characterization, mainly for surface marker expression, and basic principles of p-SC isolation and culture (mechanical separation or enzymatic digestion of the tissues, the most used culture media, cell plating conditions) will be presented. In addition, some preclinical studies that were performed in different medical areas will be cited, focusing on neurological, liver, pancreatic, heart, muscle, pulmonary, and bone diseases and also in tissue engineering field. Finally, some challenges for stem cell therapy applications will be highlighted. The understanding of the mechanisms involved in the p-SCs differentiation and the achievement of pure cell populations (after differentiation) are key points that must be clarified before bringing the preclinical studies, performed at the bench, to the medical practice.     

Clonal expansion of adult rat hepatic stem cell lines by suppression of asymmetric cell kinetics (SACK)

Adult stem cells have potential use for several biomedical applications, including cell replacement therapy, gene therapy, and tissue engineering. However, such applications have been limited due to difficulties encountered in expanding functional adult stem cells. We have developed a new approach to the problem of adult stem cell expansion based on the suppression of asymmetric cell kinetics (SACK). We postulated that asymmetric cell kinetics, required for adult stem cell function, were a major barrier to their expansion in culture. As such, conversion of adult stem cells from asymmetric cell kinetics to symmetric cell kinetics would promote their exponential expansion and longterm propagation in culture. The purine nucleoside xanthosine (Xs), which promotes guanine ribonucleotide biosynthesis, can be used to reversibly convert cells from asymmetric cell kinetics to symmetric cell kinetics. We used Xs supplementation to derive clonal epithelial cell lines from adult rat liver that have properties of adult hepatic stem cells. The properties of two Xs-derived cell lines, Lig-8 and Lig-13, are described in detail and compared to properties of adult rat hepatic cell lines derived without Xs supplementation. The Xs-derived cell lines exhibit Xs-dependent asymmetric cell kinetics and Xs-dependent expression of mature hepatic differentiation markers. Interestingly, Lig-8 cells produce progeny with properties consistent with hepatocyte differentiation, while Lig-13 progeny cells have properties consistent with bile duct epithelium differentiation. A stable adult cholangiocyte stem cell line has not been previously described. Consistent with the principles of their derivation, the SACK-derived hepatic cell lines exhibit neither senescence nor tumorigenic properties, and their differentiation properties are stable after longterm culture. These characteristics of SACK-derived stem cell lines underscore asymmetric cell kinetics as an essential adult stem cell property with potential to be the basis for a general approach to expansion and propagation of diverse adult stem cells.     

Human breast milk is a rich source of multipotent mesenchymal stem cells

Putative stem cells have been isolated from various tissue fluids such as synovial fluid, amniotic fluid, menstrual blood, etc. Recently the presence of nestin positive putative mammary stem cells has been reported in human breast milk. However, it is not clear whether they demonstrate multipotent nature. Since human breast milk is a non-invasive source of mammary stem cells, we were interested in examining the nature of these stem cells. In this pursuit, we could succeed in isolating and expanding a mesenchymal stem cell-like population from human breast milk. These cultured cells were examined by immunofluorescent labeling and found positive for mesenchymal stem cell surface markers CD44, CD29, SCA-1 and negative for CD33, CD34, CD45, CD73 confirming their identity as mesenchymal stem cells. Cytoskeletal protein marker analysis revealed that these cells expressed mesenchymal stem cells markers, namely, nestin, vimentin, smooth muscle actin and also manifests presence of E-Cadherin, an epithelial to mesenchymal transition marker in their early passages. Further we tested the multipotent differentiation potential of these cells and found that they can differentiate into adipogenic, chondrogenic and oesteogenic lineage under the influence of specific differentiation cocktails. This means that these mesenchymal stem cells isolated from human breast milk could potentially be “reprogrammed” to form many types of human tissues. The presence of multipotent stem cells in human milk suggests that breast milk could be an alternative source of stem cells for autologous stem cell therapy although the significance of these cells needs to be determined.     

成体干细胞的研究及潜在应用

成体干细胞(adultstemcells)存在于人和哺乳动物的多种成体中,具有自我更新和一定的分化潜能.现已从骨髓、软骨、血液、神经、肌肉、脂肪、皮肤、角膜缘、肝脏、胰腺等许多组织中获得干细胞,并在部分成体干细胞的体外分离培养、扩增及诱导分化等研究中取得突破性进展,发现部分成体干细胞具有预想不到的分化潜能.成体干细胞不仅是发育生物学研究的理想模型,而且是细胞移植治疗、人工组织或器官构建的种子细胞和基因治疗的理想载体细胞,因此,在揭示生命的本质和规律及再生医学中有十分广阔的应用前景.     

Transplantation-potential-related biological properties of decidua basalis mesenchymal stem cells from maternal human term placenta

Human placental decidua basalis originates from the maternal side of the placenta and has been described as a source of mesenchymal stem cells (MSCs). However, for its application in tissue regeneration and repair, the transplantation-potential-related biological properties of decidua-basalis-derived mesenchymal stem cells (DBMSCs) remain to be elucidated. We obtained DBMSCs through enzymatic digestion and density gradient centrifugation and confirmed their capacity to differentiate into cell types of the mesodermal lineage, such as osteoblasts, adipocytes and chondroblasts. Karyotype analysis showed that the isolated DBMSCs maintained chromosomal stability after long-term culture in vitro. Growth kinetics and ultrastructural observation revealed a high level of DBMSC proliferative activity. In addition, DBMSCs showed immunosuppressive properties by suppressing both mitogen- and alloantigen-induced peripheral lymphocyte proliferation. All of these properties suggest that DBMSCs, which are abundant and easily accessible, are a novel potential source of seed cells for cell transplantation treatments.