Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains

Stem cells are a valuable resource for treating disease, but limited access to stem cells from tissues such as brain restricts their utility. Here, we injected marrow stromal cells (MSCs) into the lateral ventricle of neonatal mice and asked whether these multipotential mesenchymal progenitors from bone marrow can adopt neural cell fates when exposed to the brain microenvironment. By 12 days postinjection, MSCs migrated throughout the forebrain and cerebellum without disruption to the host brain architecture. Some MSCs within the striatum and the molecular layer of the hippocampus expressed glial fibrillary acidic protein and, therefore, differentiated into mature astrocytes. MSCs also populated neuron rich regions including the Islands of Calleja, the olfactory bulb, and the internal granular layer of the cerebellum. A large number of MSCs also were found within the external granular layer of the cerebellum. In addition, neurofilament positive donor cells were found within the reticular formation of the brain stem, suggesting that MSCs also may have differentiated into neurons. Therefore, MSCs are capable of producing differentiated progeny of a different dermal origin after implantation into neonatal mouse brains. These results suggest that MSCs are potentially useful as vectors for treating a variety of central nervous system disorders.     

Seeding of recipient bone marrow cells reduces neointimal hyperplasia of de-endothelialized rat aortic allograft

Transplant vasculopathy is a leading cause of graft failure and a major contributor to the lack of success with small caliber vascular allografts. In this study we evaluate techniques of bone marrow cell seeding on small caliber vessels and assess the impact of this tactic on neointimal hyperplasia in de-endothelialized rat aortic allografts. In a preliminary study, bone marrow cells from Lewis rats were seeded onto the chemically de-endothelialized luminal surface of the abdominal aorta of WKY rats - with or without fibrin glue. In the allograft transplantation model, de-endothelialized fresh aortic allografts of WKY rats were orthotopically transplanted into Lewis recipients either directly (n = 6) or after recipient bone marrow cell seeding (n = 6). Histological evaluation was performed at 28 days. Bone marrow cells were able to adhere to the de-endothelialized aortic wall owing to the use of fibrin glue, but were unable to do so without fibrin glue. In the de-endothelialized allograft transplantation model, recipient bone marrow seeding led to a significant reduction of the ratio of intimal to medial area (0.40 +/- 0.08 versus 0.79 +/- 0.08, P = 0.0077). Some of the seeded cells remained in the intima for 4 weeks and some infiltrated the media, expressing CD31 or alpha-SMA. The results suggest that recipient bone marrow cell seeding on de-endothelialized aortic allograft is feasible with the use of fibrin glue and that this technique reduces neointimal hyperplasia of the graft.     

Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo

OBJECTIVE: Mesenchymal stem cells (MSCs), multipotential cells that reside within the bone marrow, can be induced to differentiate into various components of the marrow microenvironment, such as bone, adipose, and stromal tissues. The bone marrow microenvironment is vital to the development, differentiation, and regulation of the lymphohematopoietic system. We hypothesized that the activities of MSCs in the bone marrow microenvironment might also include immunomodulatory effects on lymphocytes. METHODS: Baboon MSCs were tested in vitro for their ability to elicit a proliferative response from allogeneic lymphocytes, to inhibit an ongoing allogeneic response, and to inhibit a proliferative response to potent T-cell mitogens. In vivo effects were tested by intravenous administration of donor MSCs to MHC-mismatched recipient baboons prior to placement of autologous, donor, and third-party skin grafts. RESULTS: MSCs failed to elicit a proliferative response from allogeneic lymphocytes. MSCs added into a mixed lymphocyte reaction, either on day 0 or on day 3, or to mitogen-stimulated lymphocytes, led to a greater than 50% reduction in proliferative activity. This effect could be maximized by escalating the dose of MSCs and could be reduced with the addition of exogenous IL-2. In vivo administration of MSCs led to prolonged skin graft survival when compared to control animals: 11.3 +/- 0.3 vs 7 +/- 0. CONCLUSIONS: Baboon MSCs have been observed to alter lymphocyte reactivity to allogeneic target cells and tissues. These immunoregulatory features may prove useful in future applications of tissue regeneration and stem cell engineering.     

心肌联合注射纤维蛋白封闭剂在细胞移植中应用的研究

目的:探讨心肌注射骨髓间充质干细胞(mesenchymal stemcells,MSCs)联合应用纤维蛋白封闭剂(FibrinSealant,FS),对细胞滞留量的影响。方法:(1)提取、分离、培养、扩增小型猪骨髓MSCs备用;(2)小型猪随机分为2组,成功建立急性心肌梗塞模型,然后分为单纯MSCs移植组和MSCs联合纤维蛋白封闭剂移植组,对比移植6周后细胞的滞留量及新生血管的密度,超声学评价心脏功能。结果:6周后MSCs FS组荧光标记的MSCs数量、新生血管密度均较MSCs组显著增多(P<0.01),MSCs FS组左室短轴缩短率(FS%)、心肌射血分数(EF%)较MSCs组显著提高(P<0.05,<0.01)。结论:经心外膜心肌注射MSCs联合纤维蛋白封闭剂较单纯心肌注射MSCs能够明显提高移植细胞的滞留存活数量,增加新生血管密度,改善心肌收缩功能。     

Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice

Stem/progenitor cells from bone marrow and other sources have been shown to repair injured tissues by differentiating into tissue-specific phenotypes, by secreting chemokines, and, in part, by cell fusion. Here we prepared the stem/progenitor cells from human bone marrow (MSCs) and implanted athem into the dentate gyrus of the hippocampus of immunodeficient mice. The implanted human MSCs markedly increased the proliferation of endogenous neural stem cells that expressed the stem cell marker Sox2. Labeling of the mice with BrdUrd demonstrated that, 7 days after implantation of the human MSCs, BrdUrd-labeled endogenous cells migrated throughout the dorsal hippocampus (positive for doublecortin) and expressed markers for astrocytes and for neural or oligodendrocyte progenitors. Subpopulations of BrdUrd-labeled cells exhibited short cytoplasmic processes immunoreactive for nerve growth factor and VEGF. By 30 days after implantation, the newly generated cells expressed markers for more mature neurons and astrocytes. Also, subpopulations of BrdUrd-labeled cells exhibited elaborate processes immunoreactive for ciliary neurotrophic factor, neurotrophin-4/5, nerve growth factor, or VEGF. Therefore, implantation of human MSCs stimulated proliferation, migration, and differentiation of the endogenous neural stem cells that survived as differentiated neural cells. The results provide a paradigm to explain recent observations in which MSCs or related stem/progenitor cells were found to produce improvements in disease models even though a limited number of the cells engrafted.     

Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms

Transplantation of whole bone marrow (BMT) as well as ex vivo-expanded mesenchymal stromal cells (MSCs) leads to striking clinical benefits in children with osteogenesis imperfecta (OI); however, the underlying mechanism of these cell therapies has not been elucidated. Here, we show that non-(plastic)-adherent bone marrow cells (NABMCs) are more potent osteoprogenitors than MSCs in mice. Translating these findings to the clinic, a T cell-depleted marrow mononuclear cell boost (> 99.99% NABMC) given to children with OI who had previously undergone BMT resulted in marked growth acceleration in a subset of patients, unambiguously indicating the therapeutic potential of bone marrow cells for these patients. Then, in a murine model of OI, we demonstrated that as the donor NABMCs differentiate to osteoblasts, they contribute normal collagen to the bone matrix. In contrast, MSCs do not substantially engraft in bone, but secrete a soluble mediator that indirectly stimulates growth, data which provide the underlying mechanism of our prior clinical trial of MSC therapy for children with OI. Collectively, our data indicate that both NABMCs and MSCs constitute effective cell therapy for OI, but exert their clinical impact by different, complementary mechanisms. The study is registered at www.clinicaltrials.gov as NCT00187018.     

cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo

Tissue engineering of large bone defects is approached through implantation of autologous osteogenic cells, generally referred to as multipotent stromal cells or mesenchymal stem cells (MSCs). Animal-derived MSCs successfully bridge large bone defects, but models for ectopic bone formation as well as recent clinical trials demonstrate that bone formation by human MSCs (hMSCs) is inadequate. The expansion phase presents an attractive window to direct hMSCs by pharmacological manipulation, even though no profound effect on bone formation in vivo has been described so far using this approach. We report that activation of protein kinase A elicits an immediate response through induction of genes such as ID2 and FosB, followed by sustained secretion of bone-related cytokines such as BMP-2, IGF-1, and IL-11. As a consequence, PKA activation results in robust in vivo bone formation by hMSCs derived from orthopedic patients.     

Chemokines in mesenchymal stem cell therapy for bone repair: a novel concept of recruiting mesenchymal stem cells and the possible cell sources

Skeletal injury is one of the most prevalent clinical problems that jeopardize the activities of daily life, especially in our aging society. Mesenchymal stem cells (MSCs) play pivotal roles in regenerating bones after bone injury. MSCs come from the surrounding tissues and/or circulation. Cell sources may be the bone marrow, periosteum, vessel walls, muscle, circulation, and elsewhere, and the migration of MSCs is necessary for bone healing. The mechanism(s) of recruitment and crucial molecules for cell migration are still unclear, but chemokines and their receptors seem to play critical roles. The induction of MSC recruitment from surrounding tissues or from the circulation can be a helpful modality to induce or to support cell-based therapy for bone regeneration.     

One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues

Most recent evidence suggests that the process of tissue repair is driven by stem-like cells that reside in multiple tissues but are replenished by precursor cells from bone marrow. Among the candidates for the reparative cells are the adult stem cells from bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). We recently found that after MSCs were replated at very low densities to generate single-cell-derived colonies, they did not exit a prolonged lag period until they synthesized and secreted considerable quantities of Dickkopf-1, an inhibitor of the canonical Wnt signaling pathway. We also found that when the cells were cocultured with heat-shocked pulmonary epithelial cells, they differentiated into epithelial cells. Most of the MSCs differentiated without evidence of cell fusion but up to one-quarter underwent cell fusion with the epithelial cells. A few also underwent nuclear fusion. The results are consistent with the interesting possibility that MSCs and similar cells repair tissue injury by three different mechanisms: creation of a milieu that enhances regeneration of endogenous cells, transdifferentiation, and perhaps cell fusion.     

From the Cover: Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury

Previous reports demonstrated that adult stem/progenitor cells from bone marrow (multipotent mesenchymal stem cells; MSCs) can repair injured tissues with little evidence of engraftment or differentiation. In exploring this phenomenon, our group has recently discovered that the therapeutic benefits of MSCs are in part explained by the cells being activated by signals from injured tissues to express an anti-inflammatory protein TNF-α–stimulated gene/protein 6 (TSG-6). Therefore, we elected to test the hypothesis that TSG-6 would have therapeutic effects in inflammatory but noninfectious diseases of the corneal surface. We produced a chemical and mechanical injury of the cornea in rats by brief application of 100% ethanol followed by mechanical debridement of corneal and limbal epithelium. Recombinant human TSG-6 or PBS solution was then injected into the anterior chamber of the eye. TSG-6 markedly decreased corneal opacity, neovascularization, and neutrophil infiltration. The levels of proinflammatory cytokines, chemokines, and matrix metalloproteinases were also decreased. The data indicated that TSG-6, a therapeutic protein produced by MSCs in response to injury signals, can protect the corneal surface from the excessive inflammatory response following injury.     

Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source

OBJECTIVE: To compare the properties of human mesenchymal stem cells (MSCs) isolated from bone marrow, synovium, periosteum, skeletal muscle, and adipose tissue. METHODS: Human mesenchymal tissues were obtained from 8 donors during knee surgery for ligament injury. After collagenase digestion or gradient-density separation, nucleated cells were plated at an appropriate density for expansion at the maximum rate without colony-to-colony contact. Yield, expandability, differentiation potential, and epitope profile were compared among MSCs from the 5 different tissue sources. RESULTS: Colony number per 10(3) nucleated cells was lower, and cell number per colony was higher, in bone marrow than in other mesenchymal tissues. When the cells were replated at low density every 14 days, bone marrow-, synovium-, and periosteum-derived cells retained their proliferation ability even at passage 10. In chondrogenesis studies in which the cells were pelleted and cultured in vitro, pellets from bone marrow-, synovium-, and periosteum-derived cells were shown to be larger and stained more extensively for cartilage matrix. Synovium-derived cells, in particular, had the greatest ability for chondrogenesis. In adipogenesis experiments, the frequency of oil red O-positive colonies was highest in synovium- and adipose tissue-derived cells. In studies of osteogenesis, the rate of alizarin red-positive colonies was highest in bone marrow-, synovium-, and periosteum-derived cells. For epitope profiling, 15 surface antigens were measured. Most appeared to have similar epitope profiles irrespective of cell source. CONCLUSION: Our findings indicate that there are significant differences in MSC properties according to tissue source, beyond donor and experimental variation. Superiority of synovium as a potential source of MSCs for clinical applications was demonstrated.     

Improvement of musculoskeletal function by cell-based therapy using mesenchymal stem cells with a prolonged life span

Cell transplantation has recently been attempted to improve musculoskeletal function. Many types of cells, such as embryonic stem cells, fetal cardiomyocytes, myoblasts, bone marrow hematopoietic cells, and mesenchymal stem cells (MSCs), have been transplanted to functionally restore damaged or diseased tissue in animal models, and marrow-derived mononuclear cells have been injected into ischemic limb clinically. MSCs can be a useful source of cell transplantation for several reasons:they have the ability to proliferate and differentiate into mesodermal tissues, including myocytes, they entail no ethical or immunological problems, and bone marrow aspiration is an established routine procedure. When placed in appropriate in vitro and in vivo environments, MSCs can give rise to all major mesenchymal tissues, such as bone, cartilage, muscle, and adipose tissue. Direct injection of murine and porcine MSCs into skeletal muscles has been shown to be feasible in murine models of ischemic limb. Large numbers of cells must be injected into damaged sites in ischemic limb to restore muscular function in humans, and cells need to be injected into the entire limb. Until now, however, there have been no reports of a sufficient number of differentiated human myocytes ever having been obtained to restore muscular function of ischemic limb. One of the reasons for this is that the life span of human cells in vitro is limited. Human cells reach senescence or stop cell growth after a limited number of cell replications, and the average number of hMSC population doublings (PDs) has been found to be 38, implying that it would be difficult to obtain enough cells to restore the function of ischemic limb. To resolve these problems and to establish a model of cell-based therapy, prolongation of the life span of hMSCs without affecting differentiation capability is essential.     

大鼠骨髓间充质干细胞培养4周膜电流研究

目的:研究未经诱导SD大鼠骨髓间充质干细胞(MSCs)培养4周(4～6代)膜电流特性,为移植细胞的选取和细胞移植后心肌电生理研究奠定基础.方法:按文献方法获得和培养MSCs至第4周,利用全细胞膜片钳技术对培养4周的MSCs膜电流进行检测,以各种钾钠钙通道阻滞剂对电流成分进行分析鉴定.结果:本实验成功封接并有电流表达细胞共39例,所有检测到的电流均以相应阻滞剂证实.共有4种钾电流表达即缓慢激活延迟整流钾电流、瞬时外向钾电流、超速激活延迟整流钾电流和内向整流钾电流(Ikir),3种外向钾电流在+60 mV时电流峰值均值分别为(0.3894±0.1230)nA、(0.4135±0.1029)nA、(0.2570±0.1135)nA;Ikir在-120mV 时电流大小为(0.9613±0.1484)nA;此外在极少数MSCs上检测到河豚毒素敏感的内向钠电流(ITTX.Na),未检测到内向钙电流.结论:培养4周后的MSCs存在钾钠电流的复合表达,从电生理角度看,MSCs有分化成功能性心肌样细胞的离子基础,其可作为体内移植较理想细胞选择.     

骨质疏松与间充质干细胞

骨髓间充质干细胞（MSCs）具有自我更新和多向分化潜能,在成年人的骨骼中它主要分化为成骨细胞和脂肪细胞。随着年龄增长,MSCs发生老化,其细胞分化方向改变,致使成骨细胞生成减少,脂肪细胞生成增多,这可能是骨质疏松（OP）的原因之一。通过移植健康的MSCs重建造骨能力,将为OP的防治提供新的思路和方法。     

In vitro analysis of multipotent mesenchymal stromal cells as potential cellular therapeutics in neurometabolic diseases in pediatric patients

Multipotent mesenchymal stromal cells (MSCs) play an important role in stromal support for hematopoietic stem cells, immune modulation, and tissue regeneration. We investigated their potential as cellular therapeutic tools in neurometabolic diseases as a growing number of affected children undergo to bone marrow transplantation. MSCs were isolated from bone marrow aspirates and expanded ex vivo under various culture conditions. MSCs under optimal good medical practice (GMP)-conform culture conditions showed the typical morphology, immunophenotype, and plasticity. Biochemically, the activities of beta-hexosaminidase A, total beta-hexosaminidase, arylsulfatase A (ASA), and beta-galactosidase measured in MSCs were comparable to those in fibroblasts of healthy donors. These four enzymes were interesting for their expression in MSCs, as each of them is defective, respectively, in well-known neurometabolic diseases. We found that MSCs released significant amounts of ASA into the media. In coculture experiments, fibroblasts from patients with metachromatic leukodystrophy, who are deficient for ASA, took up a substantial amount of ASA that was released into the media from MSCs. Mannose-6-phosphate (M6P) inhibited this uptake, which was in accordance with the M6P receptor-mediated uptake of lysosomal enzymes. Taken together, we show that MSCs produce appreciable amounts of lysosomal enzyme activities, making these cells first-choice candidates for providing metabolic correction when given to enzyme-deficient patients. With the example of ASA, it was also shown that an enzyme secreted from MSCs is taken up by enzyme-deficient patient fibroblasts. Given the plasticity of MSCs, these cells represent an interesting add-on option for cellular therapy in children undergoing bone marrow transplantation for lysosomal storage diseases and other neurometabolic diseases.     

Upregulation of AKR1C1 in mesenchymal stromal cells promotes the survival of acute myeloid leukaemia cells

The leukaemic bone marrow microenvironment, comprising abnormal mesenchymal stromal cells (MSCs), is responsible for the poor prognosis of acute myeloid leukaemia (AML). Therefore, it is essential to determine the mechanisms underlying the supportive role of MSCs in the survival of leukaemia cells. Through in silico analyses, we identified a total of 271 aberrantly expressed genes in the MSCs derived from acute myeloid leukemia (AML) patients that were associated with adipogenic differentiation, of which aldo-keto reductase 1C1 (AKR1C1) was significantly upregulated in the AML-MSCs. Knockdown of AKR1C1 in the MSCs suppressed adipogenesis and promoted osteogenesis, and inhibited the growth of co-cultured AML cell lines compared to the situation in wild- type AML-derived MSCs. Introduction of recombinant human AKR1C1 in the MSCs partially alleviated the effects of AKR1C1 knockdown. In addition, the absence of AKR1C1 reduced secretion of cytokines such as MCP-1, IL-6 and G-CSF from the MSCs, along with inactivation of STAT3 and ERK1/2 in the co-cultured AML cells. AKR1C1 is an essential factor driving the adipogenic differentiation of leukaemic MSCs and mediates its pro-survival effects on AML cells by promoting cytokine secretion and activating the downstream pathways in the AML cells.     

骨髓基质细胞治疗局灶性脑缺血的研究进展

骨髓基质细胞（MSC）是指骨髓基质中具有自我复制和多向分化潜能的干细胞,在特定条件下不仅可分化为中胚层细胞,而且也可横向分化为外胚层起源的神经胶质细胞和神经元。缺血性脑损伤时,MSC可向缺血灶迁移并分化为神经细胞,从而减轻神经功能缺损。研究表明,MSC静脉移植促进脑缺血神经功能恢复并非由于移植后新分化的神经元与宿主神经环路发生整合,而是MSC分泌的各种生长因子介导的。MSC并不能取代损伤组织,而是增进其功能,提高残存组织的可塑性。另外,MSC还是外源基因转染和表达的良好载体。MSC具有很强的增殖能力,易于体外培养扩增,通过基因修饰MSC,可提高对缺血性脑损伤的修复作用。因此,MSC有望成为基因治疗的靶细胞,在基因工程方面有着广阔的应用前景。     

Mesenchymal stromal cells transplantation in acute and chronic pancreatitis in rats

Before using MSC transplantation in the clinic to conduct preclinical studies MSCs to animals with acute and chronic pancreatitis. Work out the timing and dose of MSCs. The rationale of MSCs transplantation for the regeneration of damaged pancreatic tissue. The essence of the experiments is to establish the existence of common pathogenetic mechanisms for the development of pathological processes and sanogenesis toxic damage of pancreatic tissue. The study was work out in the rat model of acute and chronic pancreatitis, to explore beneficial and adverse effects of allogeneic stem cells for regenerative-reduction processes. For cell transplantation using allogenic stromal cell fraction of bone marrow, the cell suspension was injected at a dose of 2 x 10(6) and 5 x 10(6) cells.     

Equal modulation of endothelial cell function by four distinct tissue-specific mesenchymal stem cells

Mesenchymal stem cells (MSCs) can generate multiple end-stage mesenchymal cell types and constitute a promising population of cells for regenerative therapies. Additionally, there is increasing evidence supporting other trophic activities of MSCs, including the ability to enable formation of vasculature in vivo. Although MSCs were originally isolated from the bone marrow, the presence of these cells in the stromal vascular fraction of multiple adult tissues has been recently recognized. However, it is unknown whether the capacity to modulate vasculogenesis is ubiquitous to all MSCs regardless of their tissue of origin. Here, we demonstrated that tissue-resident MSCs isolated from four distinct tissues have equal capacity to modulate endothelial cell function, including formation of vascular networks in vivo. MSCs were isolated from four murine tissues, including bone marrow, white adipose tissue, skeletal muscle, and myocardium. In culture, all four MSC populations secreted a plethora of pro-angiogenic factors that unequivocally induced proliferation, migration, and tube formation of endothelial colony-forming cells (ECFCs). In vivo, co-implantation of MSCs with ECFCs into mice generated an extensive network of blood vessels with ECFCs specifically lining the lumens and MSCs occupying perivascular positions. Importantly, there were no differences among all four MSCs evaluated. Our studies suggest that the capacity to modulate the formation of vasculature is a ubiquitous property of all MSCs, irrespective of their original anatomical location. These results validate multiple tissues as potential sources of MSCs for future cell-based vascular therapies.