Stem and progenitor cells for neurological repair: minor issues, major hurdles, and exciting opportunities for paracrine-based therapeutics

The transplantation of cultured stem and progenitor cells is a key element in the rapidly growing field of regenerative medicine. Based on their ability to rescue and/or repair injured tissue and partially restore organ function, multiple types of stem/progenitor cells have already entered into clinical trials. However, despite several decades of intense research, the goal to apply culture-expanded stem/progenitor cells in a manner that can effectively replace cells after injury has yet to be realized. Many sources of potentially useful cells are available, but something is clearly missing. In addition, recent studies suggest that paracrine effects of secreted or released factors are responsible for most of the benefits observed after cell transplantation, rather than direct cell replacement. These data call into question the need for cell transplantation for many types of therapy, in particular for acute injuries such as myocardial infarction and stroke. In this review, we examine current progress in the area of cell transplantation and minor issues and major hurdles regarding the clinical application of different cell types. We discuss the "paracrine hypothesis" for the action of transplanted stem/progenitor cells as an opportunity to identify defined combinations of biomolecules to rescue and/or repair tissues after injury. Although many of the concepts in this review will apply to multiple injury/repair systems, we will focus primarily on stem/progenitor cell-based treatments for neurological disorders and stroke.     

骨髓干细胞移植治疗肝硬化的基础与临床研究现状

肝硬化是一种临床常见的肝病良性终末期表现。目前临床上尚缺乏有效的治疗措施。肝脏移植是最理想的治疗方法,但受供体肝脏来源限制,且费用昂贵。近年来开展的自体骨髓干细胞(BMSCs)移植治疗,为肝硬化的治疗带来了新的希望。BMSCs主要包括造型血干细胞和间充质干细胞,其具有可塑性,体外通过生长因子,体内利用特定微环境均可诱导BMSCs分化为肝前体细胞和成熟肝细胞,并明显改善肝功能。从动物实验到临床研究亦表明,BMSCs具有来源丰富、费用低廉、损伤小、自体移植不栓塞、无排斥反应等优点,为治疗肝病带来了新思路,有望成为生物人工肝的细胞来源。本文就BMSCs移植治疗肝硬化的研究现状,尤其是移植途径以及在肝脏内定居、迁移和分化机制的示踪观察方法和存在的问题作一综述,以期为从事肝病研究的同仁提供参考依据。通过对BMSCs移植从基础研究及临床应用的最新进展的描述,展示BMSCs在肝硬化治疗方面良好的治疗前景。     

间充质干细胞的细胞成像技术比较与应用

间充质干细胞是一类具有强大增殖、多向分化潜能和免疫调节能力的多功能细胞,研究显示间充质干细胞移植可能治疗多种难治性疾病,例如帕金森病、脊髓损伤以及肿瘤等。但是,人们对移植后的细胞在宿主内的存活、分布、增殖、分化、免疫排斥反应以及成瘤特性等问题尚不清楚,所以许多疾病经过细胞移植治疗后的进展及转归情况仍难以获得确切的科学证据。而细胞成像技术(包括放射性核素成像、超声成像、磁共振成像以及光学成像)可以在体外或者体内实现对间充质干细胞实时、无创的示踪,在以间充质干细胞为研究基础的细胞移植治疗和细胞组织再生的医学领域里有着巨大的应用潜力。该文综述近十年来细胞成像技术应用于示踪间充质干细胞移植疗法的研究进展,旨在比较当下多种热门细胞成像技术的优劣,进而找寻更合适的干细胞示踪策略,为干细胞移植治疗的基础和临床研究提供进一步的理论证据支持和研究思路。     

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.     

Regeneration of inner ear cells from stem cell precursors--a future concept of hearing rehabilitation?

The use of stem cells offers new and powerful strategies for future tissue development and engineering. Common features of stem cells are both their capacity for self-renewal and the ability to differentiate into mature effector cells. Since the establishment of embryonic stem cells from early human embryos, research on and clinical application of human ES cells belong to the most controversial topics in our society. Great hopes are based upon the remarkable observation that human ES cells can be greatly expanded in vitro, and that they can differentiate into various clinically important cell types. Recent advances in the cloning of mammals by nuclear transplantation provide new concepts for autologous replacement of damaged and degenerated tissues. In contrast, somatic stem cells of the adult organism were considered to be more restricted in their developmental potential. However, recent investigations suggest that somatic stem cells may have a wider differentiation potential than previously thought. In otology, initial experiments have revealed neural stem cell survival in cochlear cell cultures and under neurotrophin influence, neural stem cells seemed to develop into a neuronal phenotype. Further studies have to be carried out to investigate the full potential of stem cells as well as the molecular mechanisms that are involved in regulating cellular identity and plasticity. Clinically, advances in stem cell biology may provide a permanent source of replacement cells for treating human diseases and could open the development of new concepts for cell and tissue regeneration for a causal treatment of chronic degenerative diseases.     

Magnetic Resonance Detection of CD34+ Cells from Umbilical Cord Blood Using a 19F Label

Impaired homing and delayed recovery upon hematopoietic stem cell transplantation (HSCT) with hematopoietic stem cells (HSC) derived from umbilical cord blood (UCB) is a major problem. Tracking transplanted cells in vivo will be helpful to detect impaired homing at an early stage and allows early interventions to improve engraftment and outcome after transplantation. In this study, we show sufficient intracellular labeling of UCB-derived CD34⁺ cells, with ¹⁹F-containing PLGA nanoparticles which were detectable with both flow cytometry and magnetic resonance spectroscopy (MRS). In addition, labeled CD34⁺ cells maintain their capacity to proliferate and differentiate, which is pivotal for successful engraftment after transplantation in vivo. These results set the stage for in vivo tracking experiments, through which the homing efficiency of transplanted cells can be studied.     

Gastrointestinal stem cells. I. Pancreatic stem cells

The transplantation of islets isolated from donor pancreas has renewed the interest in cell therapy for the treatment of diabetes. In addition, the capacity that stem cells have to differentiate into a wide variety of cell types makes their use ideal to generate beta-cells for transplantation therapies. Several studies have reported the generation of insulin-secreting cells from embryonic and adult stem cells that normalized blood glucose values when transplanted into diabetic animal models. Finally, although much work remains to be done, there is sufficient evidence to warrant continued efforts on stem cell research to cure diabetes.     

Stem cells, cell transplantation and liver repopulation

Liver transplantation is currently the only therapeutic option for patients with end-stage chronic liver disease and for severe acute liver failure. Because of limited donor availability, attention has been focused on the possibility to restore liver mass and function through cell transplantation. Stem cells are a promising source for liver repopulation after cell transplantation, but whether or not the adult mammalian liver contains hepatic stem cells is highly controversial. Part of the problem is that proliferation of mature adult hepatocytes is sufficient to regenerate the liver after two-thirds partial hepatectomy or acute toxic liver injury and participation of stem cells is not required. However, under conditions in which hepatocyte proliferation is blocked, undifferentiated epithelial cells in the periportal areas, called "oval cells", proliferate, differentiate into hepatocytes and restore liver mass. These cells are referred to as facultative liver stem cells, but they do not repopulate the normal liver after their transplantation. In contrast, epithelial cells isolated from the early fetal liver can effectively repopulate the normal liver, but they are already traversing the hepatic lineage and may not be true stem cells. Mesenchymal stem cells and embryonic stem cells can be induced to differentiate along the hepatic lineage in culture, but at present these cells are inefficient in repopulating the liver. This review will characterize these various cell types and compare the properties of these cells and the conditions under which they do or do not repopulate the liver following their transplantation.     

There is More to a Lipid than just Being a Fat: Sphingolipid-Guided Differentiation of Oligodendroglial Lineage from Embryonic Stem Cells

Dr. Robert K. Yu’s research showed for the first time that the composition of glycosphingolipids is tightly regulated during embryo development. Studies in our group showed that the glycosphingolipid precursor ceramide is also critical for stem cell differentiation and apoptosis. Our new studies suggest that ceramide and its derivative, sphingosine-1-phosphate (S1P), act synergistically on embryonic stem (ES) cell differentiation. When using neural precursor cells (NPCs) derived from ES cells for transplantation, residual pluripotent stem (rPS) cells pose a significant risk of tumor formation after stem cell transplantation. We show here that rPS cells did not express the S1P receptor S1P1, which left them vulnerable to ceramide or ceramide analog (N-oleoyl serinol or S18)-induced apoptosis. In contrast, ES cell-derived NPCs expressed S1P1 and were protected in the presence of S1P or its pro-drug analog FTY720. Consistent with previous studies, FTY720-treated NPCs differentiated predominantly toward oligodendroglial lineage as tested by the expression of the oligodendrocyte precursor cell (OPC) markers Olig2 and O4. As the consequence, a combined administration of S18 and FTY720 to differentiating ES cells eliminated rPS cells and promoted oligodendroglial differentiation. In addition, we show that this combination promoted differentiation of ES cell-derived NPCs toward oligodendroglial lineage in vivo after transplantation into mouse brain.     

诱导型多能干细胞iPSc研究现状及其移植治疗帕金森综合症应用前景

诱导多能干细胞(i PS细胞)在小鼠和人上的成功获取,使干细胞领域的研究进入了一个崭新的时代。干细胞研究是再生医学的重要组成部分,研究干细胞的最终目的是应用干细胞治疗疾病,其在疾病模型建立、药物筛选、细胞移植等方面具有极大的应用潜力。i PSCs是由体细胞诱导分化而成的"多能细胞",具备和胚胎干细胞类似的功能,既解决了ESCs的伦理障碍,又为ESCs的获得提供了一条全新的途径,具有重要的理论和应用价值。i PS细胞不仅打破了道德理论的束缚,而且在再生医学、组织工程和药物发现及评价等方面具有积极的价值。神经系统遗传性疾病发病率居各系统遗传病之首,但其发病的分子机制仍不完全清楚,运用体细胞重编程技术建立的疾病特异性诱导多能干细胞模型将有助于揭示神经系统遗传性疾病的发病机理。近几年i PS细胞最新研究成果表明,利用疾病患者i PS细胞模型已逐渐应用于帕金森氏病、老年性痴呆症、脊髓侧索硬化症、脊髓肌肉萎缩症及舞蹈症等5种常见神经性退行性疾病发病机理的研究。本文主要对i PSc的发展历程,避免病毒基因干扰诱导i PS细胞进行的优化,以及干细胞尤其是i PS细胞移植治疗帕金森病等神经系统疾病的现状及应用前景进行系统阐述与论证。     

人羊膜间充质干细胞对四氯化碳诱导小鼠肝损伤定位修复的研究

目的：观察活体染料羧基荧光素乙酰乙酸（CFSE）标记的人羊膜间充质干细胞对四氯化碳诱导小鼠肝损伤模型的定位修复情况。方法：采用胰蛋白酶-胶原酶消化法从羊膜组织中分离间充质干细胞,通过流式细胞术和免疫荧光等方法进行鉴定。模型组按浓度为20μl/g剂量的四氯化碳和橄榄油混合液诱导小鼠肝损伤,治疗组经小鼠尾静脉注射羧基荧光素乙酰乙酸标记的人羊膜间充质干细胞约1×10⁶个/ml。分别取模型组和细胞移植的治疗组小鼠眼球血和肝组织进行相关检测。结果：分离得到纯度较高的羊膜间充质干细胞;冰冻切片免疫荧光显示移植1周后细胞向小鼠受损肝组织定植,CFSE标记的人羊膜间充质干细胞呈绿色荧光;细胞移植后4周,与模型组比较,细胞移植组小鼠血清中天冬氨酸转移酶、丙氨酸氨基转移酶显著降低,而白蛋白明显升高（P< 0.01）;肝组织病理切片模型组小鼠细胞水肿,坏死灶多见,脂肪变性,可见不同程度的炎性细胞浸润;治疗组小鼠肝组织病理学改变和损伤程度有较明显改善;小鼠肝组织冰冻切片的免疫荧光显示移植4周后人羊膜间充质干细胞周围分泌血清白蛋白。结论：羧基荧光素乙酰乙酸标记的人羊膜间充质干细胞可有效改善肝组织的生理功能,为细胞定位移植治疗肝脏疾病的修复情况提供实验数据。     

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.     

Neural stem cell transplantation therapy for brain ischemic stroke: Review and perspectives

Brain ischemic stroke is one of the most common causes of death and disability, currently has no efficient therapeutic strategy in clinic. Due to irreversible functional neurons loss and neural tissue injury, stem cell transplantation may be the most promising treatment approach. Neural stem cells (NSCs) as the special type of stem cells only exist in the nervous system, can differentiate into neurons, astrocytes, and oligodendrocytes, and have the abilities to compensate insufficient endogenous nerve cells and improve the inflammatory microenvironment of cell survival. In this review, we focused on the important role of NSCs therapy for brain ischemic stroke, mainly introduced the methods of optimizing the therapeutic efficacy of NSC transplantation, such as transfection and overexpression of specific genes, pretreatment of NSCs with inflammatory factors, and co-transplantation with cytokines. Next, we discussed the potential problems of NSC transplantation which seriously limited their rapid clinical transformation and application. Finally, we expected a new research topic in the field of stem cell research. Based on the bystander effect, exosomes derived from NSCs can overcome many of the risks and difficulties associated with cell therapy. Thus, as natural seed resource of nervous system, NSCs-based cell-free treatment is a newly therapy strategy, will play more important role in treating ischemic stroke in the future.     

Therapeutic implications of cancer stem cells

Most cancers comprise a heterogenous population of cells with marked differences in their proliferative potential as well as the ability to reconstitute the tumor upon transplantation. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. In addition, dysregulation of stem cell self-renewal is a likely requirement for the development of cancer. This new model for cancer will have significant ramifications for the way we study and treat cancer. In addition, through targeting the cancer stem cell and its dysregulated self-renewal, our therapies for treating cancer are likely to improve.     

Importance of the stem cell microenvironment for ophthalmological cell-based therapy

Cell therapy is a promising treatment for diseases that are caused by cell degeneration or death. The cells for clinical transplantation are usually obtained by culturing healthy allogeneic or exogenous tissue invitro. However, for diseases of the eye, obtaining the adequate number of cells for clinical transplantation is difficult due to the small size of tissue donors and the frequent needs of long-term amplification of cells in vitro, which results in low cell viability after transplantation. In addition, the transplanted cells often develop fibrosis or degrade and have very low survival. Embryonic stem cells(ESCs) and induced pluripotent stem cells(i PS) are also promising candidates for cell therapy. Unfortunately, the differentiation of ESCs can bring immune rejection, tumorigenicity and undesired differentiated cells, limiting its clinical application. Although i PS cells can avoid the risk of immune rejection caused by ES cell differentiation post-transplantation, the low conversion rate, the risk of tumor formation and the potentially unpredictable biological changes that could occur through genetic manipulation hinder its clinical application. Thus, the desired clinical effect of cell therapy is impaired by these factors. Recent research findings recognize that the reason for low survival of the implanted cells not only depends on the seeded cells, but also on the cell microenvironment, which determines the cell survival, proliferation and even reverse differentiation. When used for cell therapy, the transplanted cells need a specific three-dimensional structure to anchor and specific extra cellular matrix components in addition to relevant cytokine signaling to transfer the required information to support their growth. These structures present in the matrix in which the stem cells reside are known as the stem cell microenvironment. The microenvironment interaction with the stem cells provides the necessary homeostasis for cell maintenance and growth. A large number of studies suggest that to explore how to reconstruct the stem cell microenvironment and strengthen its combination with the transplanted cells are key steps to successful cell therapy. In this review, we will describe the interactions of the stem cell microenvironment with the stem cells, discuss the importance of the stem cell microenvironment for cell-based therapy in ocular diseases, and introduce the progress of stem cell-basedtherapy for ocular diseases.     

Retinal stem cells in vertebrates: parallels and divergences

During the development of the nervous system, after a given number of divisions, progenitors exit the cell cycle and differentiate as neurons or glial cells. Some cells however do not obey this general rule and persist in a progenitor state. These cells, called stem cells, have the ability to self-renew and to generate different lineages. Understanding the mechanisms that allow stem cells to "resist" differentiating stimuli is currently one of the most fascinating research areas for biologists. The amphibian and fish retinas, known to contain stem cell populations, have been pioneering models for neural stem cell research. The Xenopus retina enabled the characterization of the genetic processes that occur in the path from a pluripotent stem cell to a committed progenitor to a differentiated neuron. More recently, the discovery that avian and mammalian retinas also contain stem cell populations, has contributed to the definitive view of the adult nervous system of upper vertebrates as a more dynamic and plastic structure than previously thought. This has attracted the attention of clinicians who are attempting to employ stem cells for transplantation into damaged tissue. Research in this area is promising and will represent a key instrument in the fight against blindness and retinal dystrophies. In this review, we will focus primarily on describing the main characteristics of various retinal stem cell populations, highlighting their divergences during evolution, and their potential for retinal cell transplantation. We will also give an overview of the signaling cascades that could modulate their potential and plasticity.     

Genetic selection of mouse male germline stem cells in vitro: offspring from single stem cells

Spermatogenesis originates from a small population of spermatogonial stem cells. These cells are believed to divide infinitely and support spermatogenesis throughout life in the male. In this investigation, we examined the possibility of deriving transgenic offspring from single spermatogonial stem cells. Spermatogonial stem cells were transfected in vitro with a plasmid vector containing a drug resistant gene. Stably transfected stem cell clones were isolated by in vitro drug selection; these clones were expanded and used to produce transgenic progeny following spermatogonial transplantation into infertile recipients. An average of 49% of the offspring carried the transgene, and the recipient mice continued to produce monoclonal transgenic progeny a year after transplantation. Thus, a somatic cell-based genetic approach can be used to modify and select clones of spermatogonial stem cells in a manner similar to embryonic stem cells. The feasibility of genetic selection using postnatal spermatogonial stem cells demonstrates their extensive proliferative potential and provides the opportunity to develop new methods for generating stable animal transgenics or for germline gene therapy.     

Nature of leukemic stem cells in murine myelogenous leukemia

We investigated the nature of myelogenous leukemic stem cells in mice. L-8057, a megakaryoblastic leukemia cell line used in this study, produces in vivo and in vitro colonies. By means of typical chromosomal aberrations in L-8057, one can conveniently detect the origin of the cells in each colony derived from a leukemic stem cell. Direct evidence of whether cells from each colony had leukemogenicity in recipient mice was successfully obtained by the colony transplantation assay. Both leukemic colony-forming unit-spleen (L-CFU-s) and leukemic colony-forming unit-culture (L-CFU-c) in L-8057 may have belonged to the same differentiating stage in the stem cells because of their similar radiosensitivity, although some parts of the L-CFU of L-8057 seemed to have lost their capability to regenerate L-CFU-s when the cells were plated in dishes. This leukemic stem cell preserves high self-renewal ability in vitro after 10 passages. In addition, in vitro colony formation by this leukemic cell during the above course of serial passages did not require any additional exogenous stimulators. The same sort of trials have been made on other types of leukemias. Leukemic stem cells showed remarkable variety in their response to stimulating factors and in their self-renewal activity, which suggests that they may have consisted of heterogeneous populations.     

MHC expression in a human adult stem cell line and its down-regulation by hCMV US gene transfection

Due to their unique capacity to self-renew and for multiple differentiation, stem cells are considered promising candidates for cell replacement therapy in many devastating diseases. However, studies on immune rejection, which is a major problem facing successful stem cell therapy, are rare. In this study, we examined MHC expression in the M13SV1 cell line, which has previously been shown to have stem cell properties and to be non-tumorigenic, in order to determine whether human adult stem cells might be rejected after transplantation. Our results show low expression levels of MHC class I molecules on the surface of these cells. An induction of MHC class I expression was observed when the cells were treated with IFN-gamma. Maximal induction of MHC class protein expression was observed at 48 h after treatment with concentrations above 5 ng/ml of IFN-gamma. Elevated MHC class I levels were sustained for 72 h after withdrawing IFN-gamma. Therefore, we introduced human cytomegalovirus (hCMV) US genes, which are known to be able to reduce MHC class I expression on the cell surface after infection, into M13SV1 cells. Cells transfected with the hCMV US2, US3, US6 or US11 genes exhibited a reduction (40-60%) of MHC class I expression compared with mock-transfected cells. These results suggest that human adult stem cells are capable of expressing high levels of MHC class I proteins, and thus may be rejected on transplantation unless they are modified. In addition, viral stealth mechanisms can be exploited for stem cell transplantation.     

Separation technologies for stem cell bioprocessing

Stem cells have been the focus of an intense research due to their potential in Regenerative Medicine, drug discovery, toxicology studies, as well as for fundamental studies on developmental biology and human disease mechanisms. To fully accomplish this potential, the successful application of separation processes for the isolation and purification of stem cells and stem cell‐derived cells is a crucial issue. Although separation methods have been used over the past decades for the isolation and enrichment of hematopoietic stem/progenitor cells for transplantation in hemato‐oncological settings, recent achievements in the stem cell field have created new challenges including the need for novel scalable separation processes with a higher resolution and more cost‐effective. Important examples are the need for high‐resolution methods for the separation of heterogeneous populations of multipotent adult stem cells to study their differential biological features and clinical utility, as well as for the depletion of tumorigenic cells after pluripotent stem cell differentiation. Focusing on these challenges, this review presents a critical assessment of separation processes that have been used in the stem cell field, as well as their current and potential applications. The techniques are grouped according to the fundamental principles that govern cell separation, which are defined by the main physical, biophysical, and affinity properties of cells. A special emphasis is given to novel and promising approaches such as affinity‐based methods that take advantage of the use of new ligands (e.g., aptamers, lectins), as well as to novel biophysical‐based methods requiring no cell labeling and integrated with microscale technologies. Biotechnol. Bioeng. 2012; 109: 2699–2709. © 2012 Wiley Periodicals, Inc.