Autologous bone marrow transplantation/mobilization: a potential regenerative medicine for systemic degenerative disorders and healthy living

Increased longevity is compromised for the quality of life due to prevalence of metabolic derangements and systemic degenerative disorders, as a result of lifestyle changes in modern days. In a craze to improve upon quality of life, use of pharmacological formulations aimed towards symptomatic treatment regimen is mandatory to manage variety of prevailing human ailments. However; these medicines have their own limitations and side effects, soliciting alternative therapy for better living. In light of preclinical and clinical studies on adult stem cells, bone marrow transplantation in healthy elderly individuals is likely to promote rejuvenation and prevent onset of degenerative disorders as well as cure prevailing disorders if any. Progress in unraveling pleuripotency of adult bone marrow stem cells compelled us to hypothesize that the only way to regain lost cells is to replace them with cells following the principle of 'Like begets Like' which may be achieved by autologous bone marrow transplantation/mobilization.     

干细胞治疗缺血性脑卒中的研究进展

背景：干细胞是一类种类多样,具有自我复制更新能力、多向分化潜能和高度增殖潜能的细胞,其治疗缺血性脑损伤将具有良好的前景。干细胞疗法为缺血性脑卒中的治疗提供了一种新的途径,但其机制尚不能完全明确。 目的：综述干细胞的类型及其治疗缺血性脑卒中机制的研究进展。 方法：第一作者应用计算机检索1992年1月至2012年9月PubMed数据库、中国期刊全文数据库有关干细胞分类及其移植治疗缺血性脑卒中疗效、安全性、机制方面的文章,英文检索词“stem cells,brain ischemic stroke,transplantation,treatment”;中文检索词“干细胞,缺血性脑卒中,移植,治疗”。共检索到168篇相关文献,61篇文献符合纳入标准。 结果与结论：虽然干细胞移植治疗脑卒中尚处于动物模型研究阶段,但已初步显示出其广阔的发展前景。多项干细胞移植治疗脑卒中促进功能恢复的临床Ⅰ期或Ⅱ期试验已经完成。干细胞移植治疗缺血性脑卒中的实验病例没有出现不良反应并显示出功能促进效果。干细胞移植治疗缺血性脑卒中存在的主要问题包括干细胞的来源、移植途径、干细胞在宿主体内存活及与宿主脑的整合问题、治疗的有效性以及安全性等。面对已经取得的一些机制研究和临床试验结果,如何安全而迅速地将治疗缺血性脑卒中的干细胞疗法从实验推向临床,仍然是需要努力的方向。     

Endothelial progenitor cells and cerebrovascular diseases

Identifying factors that may increase the risk of stroke and assessing if treatment of such conditions may lower that risk are important in the management of cerebrovascular disease. Tobacco smoking, poor diet, hypertension and hyperlipidemia remain the major risk factors, and treatment of these conditions has been shown to significantly reduce stroke. In recent years, research has shown that stem cells from a variety of sources can be used as a tool to study and prevent the events that lead to stroke. In this regard, a population of adult stem cells, called endothelial progenitor cells (EPCs), have been identified in peripheral blood and may play an important role in tissue vascularization and endothelium homeostasis in the adult. Most of the studies on EPCs have been carried out on patients with cardiovascular diseases; however, there is emerging evidence which suggests that the introduction or mobilization of EPCs can restore tissue vascularization even after cerebrovascular diseases (CVD), such as ischemic stroke or intracerebral haemorrhage. In this review, we discuss the present level of knowledge about the characteristics of EPCs, their possible therapeutic role in CVD and how they could alter clinical practice in the future.     

Engraftment of bone marrow-derived epithelial cells

The long-held concept that transplanted bone marrow (BM)-derived cells contribute only to cells of the hematopoietic system was challenged by data from our laboratory showing that a single male BM-derived cell could not only reconstitute the hematopoietic system of an irradiated female recipient, but could also lead to the generation of mature BM-derived epithelial cells in the liver, lung, skin, and gastrointestinal tract. Careful costaining and single-cell analyses have been used to rule out false positive cells due to inadequate detection techniques in microscopy or cell overlay. Since this initial discovery, we have sought to understand the mechanisms underlying the formation of BM-derived epithelial cells, and to evaluate their therapeutic use for gene therapy and/or tissue regeneration. Several reports have shown that donor BM-derived cells, possibly macrophages, can fuse with existing host epithelial cells to form heterokaryons that express both donor and tissue-specific markers. While this is certainly true for murine tyrosinemia models, we have used a Cre-lox system to demonstrate that fusion is not a requirement for the generation of BM-derived epithelial cells and is likely not responsible for the BM-derived epithelial cells generated after standard BM transplantation. In a proof of principal experiment for potential gene therapy applications, we have shown that autologous BM-derived cells transfected with a transgene prior to BM transplantation are able to develop into mature type-II pneumocytes that express the transgene. We also discuss future research directions in the field and the therapeutic potential of BM-derived epithelia, including ongoing work to test whether combined cell and gene therapy can be used therapeutically in preclinical mouse models of human disease.     

骨髓间充质干细胞移植联合依达拉奉抑制脑梗死后的神经元凋亡

背景：国内外多项研究证实骨髓间充质干细胞移植对脑梗死组织具有一定的神经保护作用。依达拉奉是一种新型强效小分子羟自由基清除剂,可通过清除脑梗死产生的自由基,抑制神经细胞损伤,从而起到脑保护作用。 目的：观察骨髓间充质干细胞移植联合依达拉奉对大鼠脑梗死组织水通道蛋白4、Bcl-2、脑源性神经营养因子表达的影响。 方法：选取Wistar大鼠80只,建立右侧大脑中动脉闭塞模型,随机分为对照组、骨髓间充质干细胞组、依达拉奉组和联合治疗组。建模6 h后通过尾静脉分别注入移植液,对照组注射培养液,骨髓间充质干细胞组注射骨髓间充质干细胞,依达拉奉组给予依达拉奉注射液,联合组同时注入骨髓间充质干细胞和依达拉奉注射液。分别在伤后72 h将大鼠麻醉后断头取脑,应用RT-PCR、Western Blot法检测脑组织中水通道蛋白4、Bcl-2、脑源性神经营养因子基因表达和蛋白合成变化。伤后12,24,36 h取大鼠脑组织以TUNEL法测定细胞凋亡情况。 结果与结论：RT-PCR、Western Blot结果显示,在骨髓间充质干细胞与依达拉奉联合治疗组中,Bcl-2、脑源性神经营养因子的表达明显高于骨髓间充质干细胞组、依达拉奉组及对照组(P < 0.05);而水通道蛋白4的表达低于其余各组(P < 0.05)。TUNEL测定结果显示,联合治疗组中免疫组化呈棕色的凋亡细胞明显少于单独治疗组及对照组。提示骨髓间充质干细胞移植与依达拉奉联合应用治疗大鼠脑梗死,可进一步促进损伤局部脑源性神经营养因子及Bcl-2的表达,对神经细胞凋亡具有明显的抑制作用,同时可下调水通道蛋白4水平,减轻脑水肿程度,二者联合运用的效果明显优于单独治疗组。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Modulation of neuronal stem cell differentiation by hypoxia and reactive oxygen species

Low oxygen concentrations (hypoxia) occur in several physiological and pathological cellular situations such as embryogenesis and stem cell modulation (in terms of differentiation/proliferation), or ischemic stroke and cancer. On the other side of the coin, the generation of reactive oxygen species (ROS) is tightly controlled by the cell. ROS control redox sensitive signaling pathways and thus regulate cell physiology, such as programmed cell death, inflammation and/or stem cell modulation. Herein we analyze the role of hypoxia and ROS in the modulation of neuronal differentiation focusing on: (i) in vivo neurogenesis and (ii) in vitro neuronal differentiation from neural stem/precursor cells. In vivo, hypoxia promotes neurogenesis in embryos, newborns and adults, as well as in response to noxious stimuli such as ischemia. On the other hand, oxygen and ROS also play a role in in vitro neuronal differentiation. They further impact tumor growth by influencing cell proliferation and differentiation, such as in neuroblastoma development. Therefore, manipulating hypoxia and ROS production represents a useful therapeutic tool if one needs either to enhance or to modulate neurogenesis and neuronal differentiation, such as in cell replacement or in malignant cell proliferation.     

DL-3-n-Butylphthalide protects rat bone marrow stem cells against hydrogen peroxide-induced cell death through antioxidation and activation of PI3K-Akt pathway

Bone marrow stem cells (BMSCs) have been one of the most important cell sources for cell replacement therapy (CRT) in cerebral infarction. However, long-lasting oxidative stress during stroke, which plays an important role in neuron damage, deteriorates the microenvironment for cell survival, differentiation and removal. Thus the outcome of CRT in ischemic diseases was poor. DL-3-n-Butylphthalide (NBP) has protective effects on ischemic brain tissue through multiple mechanisms and has been used for stroke treatment in China for several years. In this study, hydrogen peroxide (H(2)O(2)) was used to induce oxidative stress injury to rat bone marrow stem cells (rBMSCs), imitating the microenvironment surrounding transplanted cells in the ischemic brain in vitro. The protective effects of NBP on rBMSCs against apoptosis induced by oxidative stress were investigated. Our results indicated that NBP could protect rBMSCs against apoptosis due to antioxidative properties and modulation of PI3K/Akt pathway. NBP could be used in combination with BMSCs for the treatment of cerebral infarction by improving the oxidative stress microenvironments and cell survival, however, further studies remain warranted.     

内源性骨髓间充质干细胞与骨折微环境中的相关趋化因子

背景：研究表明,骨髓间充质干细胞定向迁移依赖于损伤局部表达趋化因子与细胞表面相应受体的相互作用。然而,哪些趋化因子在介导骨髓间充质干细胞向骨折部位定向迁移过程中起关键作用,目前尚不清楚。 目的：对内源性骨髓间充质干细胞进行示踪,观察其在骨修复中的作用;检测并筛选出骨折微环境中表达量高且与骨髓间充质干细胞趋化规律相关的因子。 方法：建立C57BL/6小鼠荧光/普通骨髓嵌合体,利用嵌合体动物模型制造胫骨骨折模型,在不同时相点检测骨折部位组织绿色荧光蛋白阳性细胞占所有细胞的比例、来源于骨髓间充质干细胞的成骨细胞占全部成骨细胞的比例,判断来源于骨髓的骨髓间充质干细胞在骨折修复中的作用;利用免疫组化等方法检测骨折部位不同时相点趋化因子的蛋白表达水平。 结果与结论：骨折局部绿色荧光蛋白阳性细胞占所有细胞比例在术后1,5,14 d分别为(3.011±0.911)%,(9.031±0.145)%,(12.064±0.145)%;来源于骨髓间充质干细胞的成骨细胞占全部成骨细胞的50%以上;骨折后各时相点微环境中基质细胞衍生因子1、集落刺激因子、肝细胞生长因子、单核细胞趋化蛋白1、间质金属蛋白酶9有不同程度的表达,粒细胞集落刺激因子无明显表达。基质细胞衍生因子1的表达量相对最高。经相关分析认为：基质细胞衍生因子1在骨折微环境中的表达与骨髓间充质干细胞趋化规律具有相关关系。结果表明骨髓内的骨髓间充质干细胞参与骨折修复并在其中起到了重要作用;基质细胞衍生因子1促进骨髓间充质干细胞的定向趋化并参与骨组织的修复。     

Functional recovery after severe CNS trauma: current perspectives for cell therapy with bone marrow stromal cells

The aim of this paper is to identify current perspectives for cell therapy applied to traumatic injuries of the central nervous system (CNS). After using diverse types of cell therapy, at present there is a growing experimental evidence that transplantation of bone marrow stromal cells (BMSC) can be useful to reverse the sequels of trauma affecting the brain and spinal cord. Although we still do not know many details about how these cells achieve their beneficial effects, the application of BMSC in humans, for brain or spinal cord repair, is beginning. An exquisite caution and strict methodological controls are needed to determine with certainty whether we can open a door of hope for many patients who currently suffer severe neurological deficits that are now supposedly irreversible.     

Atorvastatin and whisker stimulation synergistically enhance angiogenesis in the barrel cortex of rats following focal ischemia

Physical therapy can promote neurovascular plasticity and functional recovery after CNS disorders such as ischemic stroke. We have previously demonstrated that whisker stimulation promotes angiogenesis in the penumbra of the ischemic barrel cortex. The aim of this study was to examine whether atorvastatin and whisker stimulation can act synergistically in enhancing angiogenesis in the barrel cortex following ischemia. Rats were divided into sham-operation, control, atorvastatin-treatment, whisker stimulation and combination therapy groups. And they were trained continuously for 10 trials per half day until the rat fulfilled 80% correct choices for continuous thirty trials, which were considered to reach the criteria. The number was significantly less in the combination therapy group than that in the whisker stimulation group (P<0.01). Immunofluorescence was used to detect angiogenesis 14d following focal ischemia. These data showed that the combination therapy was more effective in enhancing VEGF and BDNF expression than whisker stimulation (P<0.01). Our study indicated that atorvastatin can improve the discrimination ability of whisker stimulation in rats and amplify post-ischemic angiogenesis induced by whisker stimulation, potentially via enhanced expression of VEGF and BDNF in the peri-infarct region.     

Effects of interleukin-2 and interleukin-2-activated cells on in vitro myelopoiesis.

Lymphokine-activated killer (LAK) cells from human peripheral blood mononuclear cells cultured with recombinant interleukin-2 (IL-2) have been used clinically in adoptive immunotherapy for cancer patients. To study the influence of LAK cells and IL-2 on haematopoiesis, an in vitro assay system for colony formation of granulocyte-macrophage progenitor cells (GM-CFC) was used. LAK cells from cultures of either human peripheral blood (PB) or human bone marrow (BM) mononuclear cells were both inhibitory to allogeneic BM-derived GM-CFC. Inhibitory activity could be transferred with supernatants from co-cultures of LAK cells and BM targets, but also from the IL-2 activated PB- or BM-derived cells alone. The inhibitory activity from the initially non-cytotoxic/non-inhibitory BM population was rapidly induced by IL-2 activation, and preceded the generation of cytotoxic LAK cells in the culture. These experiments show that inhibition of haematopoietic progenitor cells by IL-2 is not dependent on generation of cytotoxic LAK cells, but rather the result of IL-2-induced cytokine production. We conclude that the synergistic action of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) may contribute to inhibition, but that also other cytokines are responsible for the observed inhibition of BM-derived GM-CFC.     

糖尿病对内皮祖细胞移植促血管新生作用的影响

背景：内皮祖细胞为血管新生的前体细胞,通过促血管新生作用治疗糖尿病血管病变有着良好的前景。 目的：探讨糖尿病对内皮祖细胞移植治疗缺血性疾病过程中促血管新生作用的影响。 方法：制备糖尿病大鼠模型,提取糖尿病和正常大鼠供体骨髓单个核细胞体外定向培养为内皮祖细胞。同时建立糖尿病及正常大鼠下肢缺血模型,并于缺血病变部位局部移植糖尿病或正常大鼠内皮祖细胞或PBS进行对照。移植后定期应用ELISA方法检测病变部位血管内皮生长因子含量,应用免疫组化方法计数病变部位微血管密度。 结果与结论：①受体相同,移植物不同时：移植糖尿病大鼠来源或正常大鼠来源内皮组细胞后,下肢缺血组织中血管内皮生长因子水平及微血管密度比较,差异无显著性意义(P > 0.05)。②移植物相同,受体不同时：正常大鼠移植内皮祖细胞后下肢缺血组织中血管内皮细胞生长因子含量和微血管密度均高于糖尿病组。说明在体外定向培养和病变部位局部注射条件下,糖尿病对骨髓来源内皮祖细胞移植促血管新生作用无明显影响,而对血管新生所处的病变部位微环境有明显影响。     

Isolation and characterization of dendritic cells from common marmosets for preclinical cell therapy studies

Dendritic cells (DCs) have important functions as modulators of immune responses, and their ability to activate T cells is of great value in cancer immunotherapy. The isolation of DCs from the peripheral blood of rhesus and African green monkeys has been reported, but the immune system in the common marmoset remains poorly characterized, although it offers many potential advantages for preclinical studies. In the present study, we devised methods, based on techniques developed for mouse and human DC preparation, for isolating DCs from three major tissue sources in the common marmoset: bone marrow (BM), spleen and peripheral blood. Each set of separated cells was analysed using the cell surface DC-associated markers CD11c, CD80, CD83, CD86 and human leucocyte antigen (HLA)-DR, all of which are antibodies against human antigens, and the cells were further characterized both functionally and morphologically as antigen-presenting cells. BM proved to be an excellent cell source for the isolation of DCs intended for preclinical studies on cell therapy, for which large quantities of cells are required. In the BM-derived CD11c(+) cell population, cells exhibiting the characteristic features of DCs were enriched, with the typical DC morphology and the abilities to undergo endocytosis, to secrete interleukin (IL)-12, and to stimulate Xenogenic T cells. Moreover, BM-derived DCs produced the neurotrophic factor NT-3, which is also found in murine splenic DCs. These results suggest that BM-derived DCs from the common marmoset may be useful for biological analysis and for preclinical studies on cell therapy for central nervous system diseases and cancer.     

间充质干细胞在肝纤维化形成中的作用

目的:肌成纤维细胞在肝纤维化形成中起着关键作用。有报道骨髓间充质干细胞可分化为肌成纤维细胞,并迁徙至损伤器官参与其纤维化形成过程。因此,本研究拟探讨骨髓间充质干细胞在肝纤维化形成中的可能作用及机制。方法:(1)以携带增强型绿色荧光蛋白(eGFP)的重组逆转录病毒感染大鼠间充质干细胞株(MSCs)Ap8c3进行标记(Ap8c3-eGFP)。(2)制作胆总管结扎(BDL)及猪血清腹腔注射(IPS)诱导的大鼠肝纤维化动物模型。(3)经大鼠尾静脉将Ap8c3-eGFP注入前述动物体内。(4)检测大鼠肝脏、骨髓中eGFP阳性(eGFP+)细胞分布及表达α平滑肌肌动蛋白(α-SMA)情况。结果:(1)静脉注射的Ap8c3-eGFP干细胞最终可出现于发生肝纤维化的大鼠肝脏及骨髓中。(2)大鼠肝脏中的eGFP+细胞同时表达α-SMA,主要分布于肝脏增生纤维束内。(3)迁徙至骨髓的Ap8c3-eGFP细胞可分化为肌成纤维细胞,表达α-SMA。结论:间充质干细胞可分化为肌成纤维细胞,在肝纤维化形成中起重要作用。     

Stem cells in the umbilical cord

Stem cells are the next frontier in medicine. Stem cells are thought to have great therapeutic and biotechnological potential. This will not only to replace damaged or dysfunctional cells, but also rescue them and/or deliver therapeutic proteins after they have been engineered to do so. Currently, ethical and scientific issues surround both embryonic and fetal stem cells and hinder their widespread implementation. In contrast, stem cells recovered postnatally from the umbilical cord, including the umbilical cord blood cells, amnion/placenta, umbilical cord vein, or umbilical cord matrix cells, are a readily available and inexpensive source of cells that are capable of forming many different cell types (i.e., they are “multipotent”). This review will focus on the umbilical cord-derived stem cells and compare those cells with adult bone marrow-derived mesenchymal stem cells.     

Reconstituting neurovascular unit with primary neural stem cells and brain microvascular endothelial cells in three‐dimensional matrix

Neurovascular dysfunction is a primary or secondary cause in the pathogenesis of several cerebrovascular and neurodegenerative disorders, including stroke. Therefore, the overall protection of the neurovascular unit (NVU) is a promising therapeutic strategy for various neurovascular diseases. However, the complexity of the NVU limits the study of the pathological mechanisms of neurovascular dysfunction. Reconstituting the in vitro NVU is important for the pathological study and drug screening of neurovascular diseases. In this study, we generated a spontaneously assembled three‐dimensional NVU (3D NVU) by employing the primary neural stem cells and brain microvascular endothelial cells in a Matrigel extracellular matrix platform. This novel model exhibits the fundamental structures and features of the NVU, including neurons, astrocytes, oligodendrocytes, vascular‐like structures, and blood–brain barrier‐like characteristics. Additionally, under oxygen‐glucose deprivation, the 3D NVU exhibits the neurovascular‐ or oxidative stress‐related pathological characteristics of cerebral ischemia and the injuries can be mitigated, respectively, by supplementing with the vascular endothelial growth factor or edaravone, which demonstrated that the availability of 3D NVU in ischemic stroke modeling. Finally, the 3D NVU promoted the angiogenesis and neurogenesis in the brain of cerebral ischemia rats. We expect that the proposed in vitro 3D NVU model will be widely used to investigate the relationships between angiogenesis and neurogenesis and to study the pathology and pharmacology of neurovascular diseases.     

Mesenchymal stromal/stem cells as potential therapy in diabetic retinopathy

Diabetic retinopathy (DR) is a multifactorial microvascular disease induced by hyperglycemia and subsequent metabolic abnormalities. The resulting cell stress causes a sequela of events that ultimately can lead to severe vision impairment and blindness. The early stages are characterized by activation of glia and loss of pericytes, endothelial cells (EC) and neuronal cells. The integrity of the retinal microvasculature becomes affected, and, as a possible late response, macular edema may develop as a common reason for vision loss in patients with non-proliferative DR. Moreover, the local ischemia can trigger vasoproliferation leading to vision-threating proliferative DR (PDR) in humans. Available treatment options include control of metabolic and hemodynamic factors. Timely intervention of advanced DR stages with laser photocoagulation, intraocular anti–vascular endothelial growth factor (VEGF) or glucocorticoid drugs can reduce vision loss.As the pathology involves cell loss of both the vascular and neuroglial compartments, cell replacement strategies by stem and progenitor cells have gained considerable interest in the past years. Compared to other disease entities, so far little is known about the efficacy and potential mode of action of cell therapy in treatment of DR. In preclinical models of DR different cell types have been applied ranging from embryonic or induced pluripotent stem cells, hematopoietic stem cells, and endothelial progenitor cells to mesenchymal stromal cells (MSC). The latter cell population can combine various modes of action (MoA), thus they are among the most intensely tested cell types in cell therapy. The aim of this review is to discuss the rationale for using MSC as potential cell therapy to treat DR. Accordingly, we will revise identified MoA of MSCs and speculate how these may support the repair of the damaged retina.