Endometrial stem cell transplantation in MPTP‐ exposed primates: an alternative cell source for treatment of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra. Cell‐replacement therapies have emerged as a promising strategy to slow down or replace neuronal loss. Compared to other stem cell types, endometrium‐derived stem cells (EDSCs) are an attractive source of stem cells for cellular therapies because of their ease of collection and vast differentiation potential. Here we demonstrate that endometrium‐derived stem cells may be transplanted into an MPTP exposed monkey model of PD. After injection into the striatum, endometrium‐derived stem cells engrafted, exhibited neuron‐like morphology, expressed tyrosine hydroxylase (TH) and increased the numbers of TH positive cells on the transplanted side and dopamine metabolite concentrations in vivo. Our results suggest that endometrium‐derived stem cells may provide a therapeutic benefit in the primate model of PD and may be used in stem cell based therapies.     

Salidroside induces rat mesenchymal stem cells to differentiate into dopaminergic neurons

Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of substantia nigra dopaminergic neurons that leads to a reduction in striatal dopamine (DA) levels. Replacing lost cells by transplanting dopaminergic neurons has potential value to repair the damaged brain. Salidroside (SD), a phenylpropanoid glycoside isolated from plant Rhodiola rosea, is neuroprotective. We examined whether salidroside can induce mesenchymal stem cells (MSCs) to differentiate into neuron‐like cells, and convert MSCs into dopamine neurons that can be applied in clinical use. Salidroside induced rMSCs to adopt a neuronal morphology, upregulated the expression of neuronal marker molecules, such as gamma neuronal enolase 2 (Eno2/NSE), microtubule‐associated protein 2 (Map2), and beta 3 class III tubulin (Tubb3/β‐tubulin III). It also increased expression of brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and nerve growth factor (NGF) mRNAs, and promoted the secretion of these growth factors. The expression of dopamine neurons markers, such as dopamine‐beta‐hydroxy (DBH), dopa decarboxylase (DDC) and tyrosine hydroxylase (TH), was significantly upregulated after treatment with salidroside for 1–12 days. DA steadily increased after treatment with salidroside for 1–6 days. Thus salidroside can induce rMSCs to differentiate into dopaminergic neurons.     

诱导多能干细胞在帕金森病治疗中的应用进展

帕金森病（Parkinson's disease, PD）是由于黑质中多巴胺能神经元（dopaminergic neurons, DAns）的病变导致多巴胺含量降低而引起的一种神经退行性疾病,其发病机制尚不明确,而且临床缺乏有效的早期诊断和治疗手段。诱导多能干细胞（induced pluripotent stem cells, iPSCs）的出现为神经系统疾病特别是神经退行性疾病的治疗带来了希望。基于iPSCs的细胞模型可以广泛开展PD发病机制的研究,同时以iPSCs来源的DAns、神经干细胞（neural stem cells, NSCs）等的细胞移植治疗,更是未来PD治疗最有希望的手段。从基于iPSCs的不同基因突变类型的细胞模型与不同分化程度的细胞移植治疗两个方面介绍诱导多能干细胞在PD研究中的进展,旨在分析诱导多能干细胞在帕金森病方面的应用及不足。     

Dopaminergic‐like cells from epigenetically reprogrammed mesenchymal stem cells

A number of recent studies have examined the ability of stem cells derived from different sources to differentiate into dopamine‐producing cells and ameliorate behavioural deficits in Parkinsonian models. Recently, using the approach of cell reprogramming by small cell‐permeable biological active compounds that involved in the regulation of chromatin structure and function, and interfere with specific cell signalling pathways that promote neural differentiation we have been able to generate neural‐like cells from human bone marrow (BM)‐derived MSCs (hMSCs). Neurally induced hMSCs (NI‐hMSCs) exhibited several neural properties and exerted beneficial therapeutic effect on tissue preservation and locomotor recovery in spinal cord injured rats. In this study, we aimed to determine whether hMSCs neuralized by this approach can generate dopaminergic (DA) neurons. Immunocytochemisty studies showed that approximately 50–60% of NI‐hMSCs expressed early and late dopaminergic marker such as Nurr‐1 and TH that was confirmed by Western blot. ELISA studies showed that NI‐hMSCs also secreted neurotrophins and dopamine. Hypoxia preconditioning prior to neural induction increased hMSCs proliferation, viability, expression TH and the secretion level of dopamine induced by ATP. Taken together, these studies demonstrated that hMSCs neurally modified by this original approach can be differentiated towards DA‐like neurons.     

Study of differentiated human umbilical cord–derived mesenchymal stem cells transplantation on rat model of advanced parkinsonism

The aim of this study was to explore the curative effect of differentiated human umbilical cord–derived mesenchymal stem cells (hUC‐MSCs) transplantation on rat of advanced Parkinson disease (PD) model. Human umbilical cord–derived mesenchymal stem cells were cultured and induced differentiation in vitro. The PD rats were established and allocated randomly into 2 groups: differentiated hUC‐MSCs groups and physiological saline groups (the control group). Rotation test and immunofluorescence double staining were done. The result showed that hUC‐MSCs could differentiate into mature dopamine neurons. Frequency of rotation was significantly less in differentiated hUC‐MSCs groups than in normal saline group. After we transplanted these cells into the unilateral lesioned substantia nigra induced by striatal injection of 6‐hydroxydopamine and performed in the medial forebrain bundle and ventral tegmental area, nigral tyrosine hydroxylase–positive cells were observed and survival of at least 2 months. In addition, transplantation of hUC‐MSCs could make an obviously therapeutic effect on PD rats.     

Cells therapy for Parkinson’s disease—so close and so far away

One of the strategies of treating Parkinson’s disease (PD) is the replacement of lost neurons in the substantia nigra with healthy dapamingergic cells. Potential sources for cells range from autologous grafts of dopamine secreting cells, fetal ventral mesencephalon tissue, to various stem cell types. Over the past quarter century, many experimental replacement therapies have been tried on PD animal models as well as human patients, yet none resulted in satisfactory outcomes that warrant wide applications. Recent progress in stem cell biology has shown that nuclear transfer embryonic stem cells (ntES) or induced pluripotent stem cells (iPS) derived cells can be used to successfully treat rodent PD models, thus solving the problem of immunorejection and paving the way for future autologous transplantations for treating PD. Meanwhile, however, post mortem analysis of patients who received fetal brain cell transplantation revealed that implanted cells are prone to degeneration just like endogenous neurons in the same pathological area, indicating long-term efficacy of cell therapy of PD needs to overcome the degenerating environment in the brain. A better understanding of neurodegeneration in the midbrain appeared to be a necessary step in developing new cell therapies in Parkinson’s disease. It is likely that future cell replacement will focus on not only ameliorating symptoms of the disease but also trying to slow the progression of the disease by either neuroprotection or restoring the micro-environment in the midbrain. Support by the National Key Basic Research and Development Program of China (Grant No. 2006CB0F0603) and Science and Technology Plan, Beijing Municipal Science & Technology Commission (Grant No. H020220010290)     

Cell Transplantation for Parkinson's Disease: Present Status

1. Parkinson's disease (PD) is a neurodegenerative disorder caused by the loss of neurons in the substantia nigra pars compacta and a striatal deficiency of dopamine. PD typically affects people in late middle age and progresses slowly. In the early stages of the disease, treatment targeting the dopaminergic network is effective. However, with disease progression, transplantation is an option for repairing and replacing missing dopaminergic neurons. 2. In this review, we evaluate the tissue grafts and cellular therapies that have and are being considered. Clinical trials were originally derived from transplants of adrenal medullary chromaffin cells and embryonic nigral dopaminergic neurons in patients with PD. 3. Recently, novel molecular and cellular treatments are being utilized in animals and these include embryonic stem cells, fetal cells from pigs, or transfected cells. In spite of new molecular techniques and some 20 years of experience, the transplantation therapy for PD has today the same problems and results as the first reports which used neural fetal tissue or adrenal grafts.     

Survival and engraftment of dopaminergic neurons manufactured by a Good Manufacturing Practice-compatible process

Background aimsWe have previously reported a Good Manufacturing Practice (GMP)-compatible process for generating authentic dopaminergic neurons in defined media from human pluripotent stem cells and determined the time point at which dopaminergic precursors/neurons (day 14 after neuronal stem cell [NSC] stage) can be frozen, shipped and thawed without compromising their viability and ability to mature in vitro. One important issue we wished to address is whether dopaminergic precursors/neurons manufactured by our GMP-compatible process can be cryopreserved and engrafted in animal Parkinson disease (PD) models.MethodsIn this study, we evaluated the efficacy of freshly prepared and cryopreserved dopaminergic neurons in the 6-hydroxydopamine-lesioned rat PD model.ResultsWe showed functional recovery up to 6 months post-transplantation in rats transplanted with our cells, whether freshly prepared or cryopreserved. In contrast, no motor improvement was observed in two control groups receiving either medium or cells at a slightly earlier stage (day 10 after NSC stage). Histologic analysis at the end point of the study (6 months post-transplantation) showed robust long-term survival of donor-derived tyrosine hydroxylase (TH)⁺ dopaminergic neurons in rats transplanted with day 14 dopaminergic neurons. Moreover, TH⁺ fibers emanated from the graft core into the surrounding host striatum. Consistent with the behavioral analysis, no or few TH⁺ neurons were detected in animals receiving day 10 cells, although human cells were present in the graft. Importantly, no tumors were detected in any grafted rats, but long-term tumorigenic studies will need to determine the safety of our products.ConclusionsDopaminergic neurons manufactured by a GMP-compatible process from human ESC survived and engrafted efficiently in the 6-OHDA PD rat model.     

Treatment of Parkinson's disease using cell transplantation

The clinical trials with intrastriatal transplantation of human fetal mesencephalic tissue, rich in dopaminergic neurons, in Parkinson''s disease (PD) patients show that cell replacement can work and in some cases induce major, long-lasting improvement. However, owing to poor tissue availability, this approach can only be applied in very few patients, and standardization is difficult, leading to wide variation in functional outcome. Stem cells and reprogrammed cells could potentially be used to produce dopaminergic neurons for transplantation. Importantly, dopaminergic neurons of the correct substantia nigra phenotype can now be generated from human embryonic stem cells in large numbers and standardized preparations, and will soon be ready for application in patients. Also, human induced pluripotent stem cell-derived dopaminergic neurons are being considered for clinical translation. Available data justify moving forward in a responsible way with these dopaminergic neurons, which should be tested, using optimal patient selection, cell preparation and transplantation procedures, in controlled clinical studies.     

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential

Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)]. Results. hSDFs, hASCs and WI38 exhibited a similar spindle‐like morphology and expressed same antigen profiles: positive for MSC markers (CD44, CD73 and CD105) and fibroblastic markers [collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], and negative for endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). We further analysed 90 stem cell‐associated gene expressions by performing real‐time PCR and found a more similar gene expression pattern between hASCs and hSDFs than between hSDFs and WI38. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs was observed to differ more than 2.5‐fold as compared with WI38. In addition, hSDFs and hASCs were able to form colonies and differentiate into adipocytes, osteoblasts and chondrocytes in vitro, but not WI38. Moreover, single cell‐derived hSDFs and hASCs obtained by clonal expansion were able to differentiate into adipocytes and osteoblasts. However, CD31 positive hECs did not show differentiation potential. Conclusions. These findings suggest that (i) so‐called commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony‐forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.     

Gutting the brain of inflammation: A key role of gut microbiome in human umbilical cord blood plasma therapy in Parkinson's disease model

Current therapies for Parkinson's disease (PD), including L‐3,4‐dihydroxyphenylalanine (L‐DOPA), and clinical trials investigating dopaminergic cell transplants, have generated mixed results with the eventual induction of dyskinetic side effects. Although human umbilical cord blood (hUCB) stem/progenitor cells present with no or minimal capacity of differentiation into mature dopaminergic neurons, their transplantation significantly attenuates parkinsonian symptoms likely via bystander effects, specifically stem cell graft‐mediated secretion of growth factors, anti‐inflammatory cytokines, or synaptic function altogether promoting brain repair. Recognizing this non‐cell replacement mechanism, we examined here the effects of intravenously transplanted combination of hUCB‐derived plasma into the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced rat model of PD. Animals received repeated dosing of either hUCB‐derived plasma or vehicle at 3, 5 and 10 days after induction into MPTP lesion, then behaviourally and immunohistochemically evaluated over 56 days post‐lesion. Compared to vehicle treatment, transplantation with hUCB‐derived plasma significantly improved motor function, gut motility and dopaminergic neuronal survival in the substantia nigra pars compacta (SNpc), which coincided with reduced pro‐inflammatory cytokines in both the SNpc and the intestinal mucosa and dampened inflammation‐associated gut microbiota. These novel data directly implicate a key pathological crosstalk between gut and brain ushering a new avenue of therapeutically targeting the gut microbiome with hUCB‐derived stem cells and plasma for PD.     

Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult‐derived neural stem cell cultures

The adult rat hippocampus contains fibroblast growth factor 2–responsive stem cells that are self‐renewing and have the ability to generate both neurons and glia in vitro, but little is known about the molecular events that regulate stem cell differentiation. Hippocampus‐derived stem cell clones were used to examine the effects of retinoic acid (RA) on neuronal differentiation. Exposure to RA caused an immediate up‐regulation of NeuroD, increased p21 expression, and concurrent exit from cell cycle. These changes were accompanied by a threefold increase in the number of cells differentiating into immature neurons. An accompanying effect of RA was to sustain or up‐regulate trkA, trkB, trkC, and p75NGFR expression. Without RA treatment, cells were minimally responsive to neurotrophins (NTs), whereas the sequential application of RA followed by brain‐derived neurotrophic factor or NT‐3 led to a significant increase in neurons displaying mature γ‐a‐minobutyric acid, acetylcholinesterase, tyrosine hydroxylase, or calbindin phenotypes. Although NTs promoted maturation, they had little effect on the total number of neurons generated, suggesting that RA and neurotrophins acted at distinct stages in neurogenesis. RA first promoted the acquisition of a neuronal fate, and NTs subsequently enhanced maturation by way of RA‐dependent expression of the Trk receptors. In combination, these sequential effects were sufficient to stimulate stem cell–derived progenitors to differentiate into neurons displaying a variety of transmitter phenotypes. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 65–81, 1999     

体外诱导人骨髓间充质干细胞向多巴胺神经元分化的研究

通过体外诱导人骨髓间充质干细胞(bone marrow mesenchymal stemcells,BMSCs)向多巴胺(dopamine,DA)神经元分化,探讨人BMSCs来源的DA神经元的功能特征及其分化机制,为临床上细胞移植替代治疗诸如帕金森氏病(parkinson’sdisease,PD)等神经精神性疾病提供一种理想的细胞来源。通过密度梯度离心获取人骨髓中的单个核细胞,贴壁培养纯化BMSCs。50μmol/L脑源性神经营养因子(brain derivedneurotrophy factor,BDNF),10μmol/Lforskolin(FSK)和10μmol/LDA联合对BMSCs进行诱导。电子显微镜观察诱导2周后细胞是否具有神经元的超微结构特点;免疫细胞化学染色和RT-PCR检测DA神经元分化过程中的标志物酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达以及转录因子Nurr1、Ptx3和Lmx1b的表达;高效液相色谱(highperformance liquid chromatogram,HPLC)检测诱导2周后的细胞多巴胺的释放水平。结果表明,诱导2周后,电镜下细胞胞浆中有大量密集的呈扁平囊状的粗面内质网及其间的一些游离核糖体以及神经微丝的形成。RT-PCR结果显示NSE(neuron specificenolase)、Nurr1、Ptx3、Lmx1b和TH的mRNA均有表达;免疫细胞化学染色结果表明诱导2周后TH阳性细胞(24·80±3·36)%的表达较诱导3d后(3·77±1·77)%明显提高(P<0·01);HPLC检测到诱导2周后的细胞DA释放水平[(1·22±0·36)μg/mL(n=6)]高于未经诱导的细胞[(0·75±0·22)μg/mL(n=6)(t=-2·79,P=0·038)]。由此得出,BDNF、FSK和DA可以在体外诱导人BMSCs向DA神经元分化,并具有DA神经元的功能特征,是临床用于治疗神经精神性疾病的理想细胞来源。     

Therapeutic benefits of CD90‐negative cardiac stromal cells in rats with a 30‐day chronic infarct

Cardiac stromal cells (CSCs) can be derived from explant cultures, and a subgroup of these cells is viewed as cardiac mesenchymal stem cells due to their expression of CD90. Here, we sought to determine the therapeutic potential of CD90‐positive and CD90‐negative CSCs in a rat model of chronic myocardial infarction. We obtain CD90‐positive and CD90‐negative fractions of CSCs from rat myocardial tissue explant cultures by magnetically activated cell sorting. In vitro, CD90‐negative CSCs outperform CD90‐positive CSCs in tube formation and cardiomyocyte functional assays. In rats with a 30‐day infarct, injection of CD90‐negative CSCs augments cardiac function in the infarct in a way superior to that from CD90‐positive CSCs and unsorted CSCs. Histological analysis revealed that CD90‐negative CSCs increase vascularization in the infarct. Our results suggest that CD90‐negative CSCs could be a development candidate as a new cell therapy product for chronic myocardial infarction.     

The differentiation of human placenta-derived mesenchymal stem cells into dopaminergic cells in vitro

Mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after Parkinson’s disease. MSCs have significant advantages over other stem cell types, and greater potential for immediate clinical application. The aim of this study was to investigate whether MSCs from the human placenta could be induced to differentiate into dopaminergic cells. MSCs from the human placenta were isolated by digestion and density gradient fractionation, and their cell surface glycoproteins were analyzed by flow cytometry. These MSCs were cultured under conditions promoting differetiation into adipocytes and osteoblasts. Using a cocktail that includes basic fibroblast growth factor (bFGF), all trans retinoic acid (RA), ascorbic acid (AA) and 3-isobutyl-1-methylxanthine (IBMX), the MSCs were induced in vitro to become dopamine (DA) neurons. Then, the expression of the mRNA for the Nestin and tyrosine hydroxylase (TH) genes was assayed via RT-PCR. The expression of the Nestin, dopamine transporter (DAT), neuronal nuclear protein (NeuN) and TH proteins was determined via immunofluorescence. The synthesized and secreted DA was determined via ELISA. We found that MSCs from the human placenta exhibited a fibroblastoid morphology. Flow cytometric analyses showed that the MSCs were positive for CD44 and CD29, and negative for CD34, CD45, CD106 and HLA-DR. Moreover, they could be induced into adipocytes and osteocytes. When the MSCs were induced with bFGF, RA, AA and IBMX, they showed a change in morphology to that of neuronal-like cells. The induced cells expressed Nestin and TH mRNA, and the Nestin, DAT, NeuN and TH proteins, and synthesized and secreted DA. Our results suggest that MSCs from the human placenta have the ability to differentiate into dopaminergic cells.     

Embryonic stem cell-derived neuron models of Parkinson's disease exhibit delayed neuronal death

Establishment of a Parkinson's disease (PD) neuron model was attempted with mouse embryonic stem (ES) cells. ES cell lines over-expressing mouse nuclear receptor-related 1 (Nurr1), together with human wild-type and alanine 30 --> proline (A30P) and alanine 53 --> threonine (A53T) mutant alpha-synuclein were established and subjected to differentiation into dopaminergic neurons. The ES cell-derived dopaminergic neurons expressing wild-type or mutant alpha-synuclein exhibited the fundamental characteristics consistent with dopaminergic neurons in the substantia nigra. The ES cell-derived PD model neurons exhibited increased susceptibility to oxidative stress, proteasome inhibition, and mitochondrial inhibition. Cell viability of PD model neurons and the control neurons was similar until 28 days after differentiation. Nonetheless, after that time, PD model neurons gradually began to undergo neuronal death over the course of 1 month, showing cytoplasmic aggregate formation and an increase of insoluble alpha-synuclein protein. Such delayed neuronal death was observed in a mutant alpha-synuclein protein level-dependent manner, which was slightly inhibited by a c-jun N-terminal kinase inhibitor and a caspase inhibitor. Such cell death was not observed when the same ES cell lines were differentiated into oligodendrocytes. The ES cell-derived PD model neurons are considered as prospective candidates for a new prototype modelling PD that would allow better investigation of the underlying neurodegenerative pathophysiology.