Generation of immunosuppressive mesenchymal stem cells in allogeneic human serum

Mesenchymal stem cells (MSC) may be used to treat acute graft-versus-host disease and for tissue repair. In vitro expansion of MSC has been achieved in the presence of fetal calf serum (FCS). For safety and regulatory reasons, we explored if FCS could be replaced by human blood group AB serum. Proliferation and fold increase of MSC was higher in the presence of AB-serum, compared to FCS. Similar to cells generated in FCS media, MSC from AB-serum media were more than 95% positive for CD90, CD105 and human leukocyte antigen (HLA) class I, and negative for hematopoietic and endothelial markers CD14, CD31, CD34, CD45, and CD80. HLA class II expression was higher in MSC generated in AB-serum, but decreased with higher passage numbers. MSC generated in AB-serum suppressed lymphocyte proliferation in mixed lymphocyte cultures and after stimulation with phytohemagglutinin. MSC expanded in AB-serum and FCS have similar in vitro properties.     

Acceleration of spinal fusion using syngeneic and allogeneic adult adipose derived stem cells in a rat model

Posterolateral spinal fusion is the standard treatment for lumbar compression fractures. Adult adipose tissue‐derived stem cells (ASCs) promote osteogenesis in vivo and in vitro. The hypothesis tested in this study was that syngeneic and allogeneic ASCs on a biomaterial scaffold composed of tricalcium phosphate and collagen I will accelerate spinal fusion in a rat model. ASCs from male Fischer or ACI rats were loaded onto scaffolds (53,571 cells/mm³) and cultured in stromal media for 48 h. Male Fisher rats were assigned to 4 cohorts (n = 14/cohort) after bilateral decortication of the L4 and L5 transverse processes: (1) No treatment; (2) scaffold only; (3) scaffold + syngeneic ASCs; or (4) scaffold + allogeneic ASCs. Half of each cohort was harvested 4 or 8 weeks after surgery. Spinal fusion was evaluated with radiographs, microcomputed tomography, and light microscopy. Callus did not form in spines without scaffolds. There were no significant differences in callus formation among scaffold cohorts 4 weeks after surgery. Callus formation was more mature in both ASC cohorts versus scaffold alone 8 weeks after surgery based on microstructure as well as radiographic and microcomputed tomographic evidence of active bone formation. Inflammatory cell infiltrate was significantly lower in both ASC cohorts (syngeneic = 18.3 ± 0.85%; allogeneic = 23.5 ± 2.33%) versus scaffold alone (46.8 ± 11.8%) 4 weeks after surgery. Results of this study support syngeneic and allogeneic ASC acceleration of posterior lumbar spinal fusion in a rat model. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:366–373, 2009     

Transplantation of adult rat spinal cord stem/progenitor cells for spinal cord injury

Stem/progenitor cells derived from the ependymal region of the spinal cord have the ability to self-renew and are multipotential for neurons and glia. These cells may have the ability to regenerate the injured mammalian spinal cord as they do in some lower vertebrates. However, the optimal conditions for transplantation and the fate of transplanted cells are not fully known. In the current study, spinal cord stem/progenitor cells were cultured from adult male rats expressing enhanced green fluorescent protein (eGFP). Neurospheres were transplanted at the time of clip compression injury (35-g force) into the injury site, or 1 mm rostral and caudal to the injury site. Neurospheres were also transplanted into a subacute model (day 9 after injury) and a chronic model (day 28 after injury). Functional recovery was also studied in an acute injury model with weekly locomotor testing over a 16-week period. A significant increase in cell survival at 7 days was seen in rats receiving rostral and caudal injections as compared to injection directly into the site of injury. A significant increase in cell survival was also seen in rats receiving subacute transplants at 9 days after injury. Transplanted cells differentiated primarily into astrocytes (31.2%) and oligodendrocytes (50.3%), and a small number of neurons (1%). No improvement was seen in the Basso, Beattie and Bresnahan (BBB) locomotor rating scale after acute transplantation as compared with injury only, although surviving transplanted cells were identified that had migrated across the injury site from the rostral and caudal injection sites.     

Cotransplantation of mouse neural stem cells (mNSCs) with adipose tissue-derived mesenchymal stem cells improves mNSC survival in a rat spinal cord injury model

The low survival rate of graft stem cells after transplantation into recipient tissue is a major obstacle for successful stem cell therapy. After transplantation into the site of spinal cord injury, the stem cells face not only hypoxia due to low oxygen conditions, but also a lack of nutrients caused by damaged tissues and poor vascular supply. To improve the survival of therapeutic stem cells after grafting into the injured spinal cord, we examined the effects of cotransplanting mouse neural stem cells (mNSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) on mNSC viability. The viability of mNSCs in coculture with AT-MSCs was significantly increased compared to mNSCs alone in an in vitro injury model using serum deprivation (SD), hydrogen peroxide (H(2)O(2)), and combined (SD + H(2)O(2)) injury mimicking the ischemic environment of the injured spinal cord. We demonstrated that AT-MSCs inhibited the apoptosis of mNSCs in SD, H(2)O(2), and combined injury models. Consistent with these in vitro results, mNSCs transplanted into rat spinal cords with AT-MSCs showed better survival rates than mNSCs transplanted alone. These findings suggest that cotransplantation of mNSCs with AT-MSCs may be a more effective transplantation protocol to improve the survival of cells transplanted into the injured spinal cord.     

Transplantation of neurotrophin-3-expressing bone mesenchymal stem cells improves recovery in a rat model of spinal cord injury

Background

This study aimed to investigate the therapeutic effects of transplanting neutrophin-3 (NT-3)-expressing bone marrow-derived mesenchymal stem cells (BMSCs) in a rat model of spinal cord injury (SCI).

Methods

Forty-eight adult female Sprague–Dawley rats were randomly assigned to three groups: the control, BMSC, and NT-3-BMSC groups. BMSCs were infected with NT-3-DsRed or DsRed lentivirus and injected into the cerebrospinal fluid (CSF) via lumbar puncture (LP) 7 days after SCI in the NT-3-BMSC and BMSC groups, respectively. The hind-limb motor function of all rats was recorded using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale on days 1, 3, 7, 14, 21, 28, and 35 after transplantation. Haematoxylin-eosin (HE) staining, immunofluorescence labelling, and western blotting were performed at the final time point.

Results

Expressions of NT-3, brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF) proteins increased significantly in the NT-3-BMSC group, and hind-limb locomotor functions improved significantly in the NT-3-BMSC group compared with the other two groups. The cystic cavity area was smallest in the NT-3-BMSC group. In the NT-3-BMSC group, neurofilament 200 (NF200) and glial fibrillary acidic protein (GFAP) expression levels around the lesions were significantly increased and decreased, respectively.

Conclusions

Our findings demonstrate that transplantation of NT-3 gene-modified BMSCs via LP can strengthen the therapeutic benefits of BMSC transplantation. We observed that these modified cells increased locomotor function recovery, promoted nerve regeneration, and improved the injured spinal cord microenvironment, suggesting that it could be a promising treatment for SCI.     

Cotransplantation of human embryonic stem cell-derived neural progenitors and schwann cells in a rat spinal cord contusion injury model elicits a distinct neurogenesis and functional recovery

Cotransplantation of neural progenitors (NPs) with Schwann cells (SCs) might be a way to overcome low rate of neuronal differentiation of NPs following transplantation in spinal cord injury (SCI) and the improvement of locomotor recovery. In this study, we initially generated NPs from human embryonic stem cells (hESCs) and investigated their potential for neuronal differentiation and functional recovery when cocultured with SCs in vitro and cotransplanted in a rat acute model of contused SCI. Cocultivation results revealed that the presence of SCs provided a consistent status for hESC-NPs and recharged their neural differentiation toward a predominantly neuronal fate. Following transplantation, a significant functional recovery was observed in all engrafted groups (NPs, SCs, NPs + SCs) relative to the vehicle and control groups. We also observed that animals receiving cotransplants established a better state as assessed with the BBB functional test. Immunohistofluorescence evaluation 5 weeks after transplantation showed invigorated neuronal differentiation and limited proliferation in the cotransplanted group when compared to the individual hESC-NP-grafted group. These findings have demonstrated that the cotransplantation of SCs with hESC-NPs could offer a synergistic effect, promoting neuronal differentiation and functional recovery.     

大鼠睾丸支持细胞对骨髓问充质干细胞免疫抑制作用的影响

目的探索睾丸支持细胞（Sertoli细胞）对骨髓间充质干细胞（BMSC）免疫抑制作用的影响,为二者在移植免疫中的联合应用提供思路。方法二步酶解法处理大鼠睾丸,分离Sertoli细胞;Percoll法分离大鼠BMSC;Ficoll法分离淋巴细胞;刀豆蛋白A（ConA）刺激进行T细胞转化试验;将Sertoli细胞、BMSC和Sertoli细胞＋BMSC分别加入未经ConA处理的静止淋巴细胞培养体系和经ConA处理后的T细胞转化体系,MTT法测定淋巴细胞增殖情况,观察BMSC、Sertoli细胞或二者共培养对T细胞活化、增殖的影响。结果 BMSC、Sertoli细胞以及二者共培养对静止的淋巴细胞无明显作用。BMSC、Sertoli细胞及二者共培养对T细胞的活化、增殖均有明显的抑制作用,且Sertoli细胞与BMSC共培养时抑制作用呈现一定的协同性。结论 BMSC和Sertoli细胞均具有负性免疫调节作用,二者共培养可以进一步增强BMSC的免疫抑制效应。     

优化肝移植术后免疫抑制治疗方案

免疫抑制剂的毒副作用常常是影响肝移植受者长期生存的危险因素.免疫抑制个体化治疗是目前肝脏移植综合治疗的热点和难点.免疫抑制治疗已从仅着眼于移植术后抗免疫排斥反应,逐步向追求患者和移植物长期存活、药物副作用最小化、优化患者生命质量,同时降低患者经济负担方向发展.通过掌握各类免疫抑制剂特点及毒副作用,正确有效地评估受者的免疫状态,结合患者自身病理生理状况,有针对性地选择免疫抑制治疗方案,达到药物剂量最小化,实现个体化给药方案.     

Immunomodulatory effects of mesenchymal stem cells in a rat organ transplant model

BACKGROUND: Recent reports suggest that mesenchymal stem cells (MSCs) have immunomodulatory properties. Mesenchymal stem cells can suppress the immune response toward alloantigen in vitro by inhibiting T cell proliferation in mixed-lymphocyte reactions (MLRs). However, relatively little has been reported regarding the immunomodulative potential of MSCs in vivo. Herein the authors confirm the immunomodulatory effects of rat MSCs in vitro and tested for tolerogenic features in a model of allogeneic heart transplantation. METHODS: Mesenchymal stem cells were obtained from bone marrow aspirates of male Lewis rats (major histocompatibility complex [MHC] haplotype RT1) and ACI rats (RT1). Lewis MSCs were cocultured with ACI spleen cells to reveal direct effects of MSCs on lymphocytes. In addition, MSCs were added to MLRs between ACI T cells as responders and irradiated Lewis spleen cells as stimulators. Finally, MSCs were administered in an allogeneic heart transplantation model at different doses (with and without cyclosporin A [CsA]). RESULTS: Mesenchymal stem cells appeared with typical spindle-shaped morphology in culture and readily differentiated into adipocytes when exposed to differentiation media. Mesenchymal stem cells expressed MHC class I, but not class II or costimulatory molecules. In vitro, MSCs phagocytosed ACI spleen cells. When introduced into an MLR, donor MSCs suppressed the proliferation of stimulated T cells. However, in vivo, MSC injection did not prolong allograft survival. In addition, concurrent treatment with low-dose CsA and MSCs accelerated allograft rejection. CONCLUSIONS: The present data confirm previous reports suggesting that MSCs have immunomodulatory properties in vitro. However, their tolerogenic properties in vivo must be questioned, as MSC injections were not only ineffective at prolonging allograft survival, but tended to promote rejection.     

A chronic renal rejection model with a fully MHC-mismatched rat strain combination under immunosuppressive therapy

BackgroundThe Fischer-to-Lewis (LEW) rat model of kidney transplantation is a widely accepted and well-characterized model of chronic rejection. In contrast to transplantation in a clinical setting, however, the absence of treatment with immunosuppressants and only minor mismatch of major histocompatibility complexes (MHCs) are critical discrepancies. Here, we established a rat model of chronic rejection using fully MHC-mismatched strains in which kidney disease progresses even under immunosuppressive therapy.MethodsLEW (RT1^l) rats were used as donors and Brown Norway (BN, RT1ⁿ) rats as recipients. Intramuscular administration of 0.1 mg/kg of tacrolimus was initiated on the day of transplantation. Post-transplantation, this dose was maintained until Day 9, suspended until Day 28 and then resumed from Day 29. Renal function, histopathology, and levels of donor-specific antibody (DSA) and several biomarkers of renal injury were assessed.ResultsOn Day 91 post-transplantation, recipients received tacrolimus treatment with short-term suspension exhibited reduced renal function and changes in histology. Those were characteristics of chronic rejection including glomerulosclerosis, interstitial fibrosis, and tubular atrophy in human transplantation recipients. Urinary protein excretion increased in a linear fashion, and elevated levels of several biomarkers of renal injury and DSA were observed even under administration of an immunosuppressant.ConclusionsWe established an allograft rejection model with impaired renal function and typical histopathological changes of chronic rejection in fully MHC-mismatched rats by controlling administration of an immunosuppressant. These findings suggest that this model more accurately reflects transplantation in a clinical setting than existing models and enables the evaluation of therapeutic agents.     

Cell therapy using human embryonic stem cells

Cell therapy refers to the transplantation of healthy, functional and propagating cells to restore the viability or function of deficient tissues. Stem cells are characterized by self-renewal and the potential to form differentiated cells. In early mammalian embryos, at the blastocyst stage, the inner cell mass is pluripotent. Thus, it has been recognized that human embryonic stem cells (hESCs), which are derived from such cells of blastocysts, may serve as a source of numerous types of differentiated cells. The first part of this review summarizes different techniques for the derivation and maintenance of undifferentiated hESCs. In the second part, issues concerning the safety and bulk production, which may enable hESCs use in future clinical applications, are presented. The last part of this review details accumulated data regarding the in vitro differentiation potential of hESCs.     

人胚神经干细胞移植治疗大鼠脊髓损伤

目的 探讨人胚神经干细胞（hNSC）移植治疗脊髓损伤（SCI）的可行性。方法 分离、培养和鉴定hNSC；用5溴-2脱氧尿苷嘧啶（BrdU）标记hNSC，并将其移植到14只T10半横断的Wistar大鼠损伤脊髓内（另外14只T10半横断损伤的大鼠作为对照组，仅损伤脊髓内注射DMEM／F12培养液），用BrdU的FITC免疫荧光染色检测移植细胞的存活和迁徙，用NF-200、GFAP免疫组织化学鉴定移植细胞的分化，BBB评分评定大鼠功能恢复情况。结果 （1）获得了大量的hNSC；（2）用免疫组织化学可以检测到移植的hNSC能在体内长时间存活（达2个月）并向远处迁徙，并分化为神经元和胶质细胞；（3）检测到实验组大鼠BBB得分明显高于对照组大鼠（P〈0．01），在SCI后第10周时实验组和对照组BBB得分最大差距达到2．1分。结论 hNSC移植能促进SCI大鼠后肢功能恢复，它是SCI移植治疗较有价值的细胞资源。     

Mechanism of spinal cord injury regeneration and the effect of human neural stem cells-secretome treatment in rat model

BACKGROUND Globally, complete neurological recovery of spinal cord injury(SCI) is still less than 1%, and 90% experience permanent disability. The key issue is that a pharmacological neuroprotective-neuroregenerative agent and SCI regeneration mechanism have not been found. The secretomes of stem cell are an emerging neurotrophic agent, but the effect of human neural stem cells(HNSCs) secretome on SCI is still unclear.AIM To investigate the regeneration mechanism of SCI and neuroprotective-neuro...     

输注供鼠骨髓间充质干细胞延长肾移植大鼠的存活时间

目的 探讨在同种大鼠肾移植中输注供者骨髓间充质干细胞(MSC)对急性排斥反应的影响以及延长受鼠存活时间的作用.方法 取Wistar大鼠骨髓,分离和培养其MSC.以Wistar 大鼠为供者,Lewis大鼠为受者,建立同种大鼠肾移植模型.根据受鼠处理方式的不同,分为低剂量MSC组、高剂量MSC组、CsA组及对照组,低剂量MSC组和高剂量MSC组于移植前后分别多次输注1×106个和t×107个供者MSC,CsA组于术后2d开始腹腔内注入CsA 0.5 mg·kg-1 ·d-1,以腹腔内注射PBS作为对照.移植后,比较各组受鼠的存活时间,观察各组移植肾功能及移植肾组织病理学改变.结果 低剂量MSC组、高剂量MSC组、CsA组及对照组受鼠的存活时间分别为(21.7±7.2)d、(31.2±14.3)d、(34.9±15.7)d及(9.0±2.3)d;低剂量MSC组、高剂量MSC组和CsA组存活时间均明显长于对照组(P＜0.01),而低剂量MSC组存活时间明显短于高剂量MSC组和CsA组(P＜0.05).术后第4天,高剂量MSC组和CsA组移植肾组织形态和结构基本正常;对照组移植肾组织则表现出典型的急性排斥反应,出现广泛间质性浸润,肾小管炎症和片状坏死、出血,肾小球炎症浸润严重;而与对照组相比,低剂量MSC组急性排斥反应的病理表现则要明显减轻.结论 同种肾移植大鼠输注供者MSC后,可以达到有效的免疫调节作用,并且可明显延长大鼠的存活时间,呈MSC剂量依赖性.     

Structured coculture of stem cells and disc cells prevent disc degeneration in a rat model

Background contextHarnessing the potential of stem cells is an important strategy for regenerative medicine. This study explores the use of bilaminar coculture pellets (BCPs) of mesenchymal stem cells (MSCs) and nucleus pulposus cells (NPCs) as a cell-based therapy for intervertebral disc regeneration. Prior in vitro experiments have shown that BCP can help differentiate MSCs and substantially improve new matrix deposition.PurposeTo evaluate the clinical relevance of BCPs by testing the system in vivo.Study design/settingWe have designed a novel spherical BCP where MSCs are enclosed in a shell of NPCs. The pellets were tested in vivo in a rat tail model of disc degeneration.MethodsRat caudal intervertebral discs were denucleated and treated with BCP in a fibrin sealant (FS) carrier (controls were MSCs suspended in FS; NPCs suspended in FS; MSCs and NPCs suspended in FS; FS only; and surgery only). At 14 and 35 days after implantation, the animals were euthanized and discs were evaluated for proteoglycan content, enzyme-linked immunosorbent assay for inflammatory cytokines, cell retention using polymerase chain reaction, disc height, histology, and disc grade based on a blinded scoring system.ResultsThe proteoglycan and cytokine levels were not significantly different among groups. The BCP group had higher cell retention than controls. Disc height and disc grade increased over time only in the BCP group. Bilaminar coculture pellets were the only treatment to show proteoglycan staining in the nucleus space at 35 days.ConclusionsThis study shows that BCPs may prevent postnucleotomy disc degeneration in vivo. Larger animals and longer time points will be necessary to further judge potential clinical impact. As opposed to strategies that require growth factor supplements, predifferentiation, or genetic manipulations, BCPs are a self-sustaining and targeted method for tissue regeneration in situ.     

Evidence of pluripotent human prostate stem cells in a human prostate primary xenograft model

INTRODUCTION: The phenotypic plasticity of the human prostate stem cell within human prostate tissue was examined to determine the response of the stem cell to changes in the androgenic environment. METHODS: Prostate xenografts were transplanted into athymic nu/nu mice implanted with testosterone pellets, allowed to establish for 1 month time point, the hosts were castrated and pellets removed, and following 1 month of androgen deprivation, the hosts were stimulated with androgen for 2 days to induce proliferation of the residual population of stem cells (2-month time point). RESULTS: Glands in benign xenografts harvested at the 1- and 2-month time points contained basal cell layers that expressed p63 and high molecular weight cytokeratin, and in which essentially all of the cellular proliferation was localized, consistent with the proposed localization of the prostate stem cell. Benign glandular structures in the xenografts were populated by basal, secretory epithelial, neuroendocrine (NE), or squamous cells overlaying the basal cell layer, whereas, adenocarcinoma glands in the xenografts resembled the original prostate cancer (CaP) tissue. CONCLUSIONS: In this human prostate primary xenograft model, the residual stem cell population that survives transplantation, or androgen deprivation, maintains significant pluripotentiality as demonstrated by the capacity to generate progeny that differentiate along multiple lineages in response to microenvironmental signals, particularly along the secretory epithelial lineage in response to androgen, and along the NE cell lineage in response to androgen deprivation.