Advances and perspectives on cellular therapy in acquired bone marrow failure diseases

Acquired bone marrow failure diseases(ABMFD) are a class of hematopoietic stem cell diseases with a commonality of non-inherited disruption of hematopoiesis that results in pancytopenia. ABMFDs also are a group of heterogeneous diseases with different etiologies and treatment options. The three most common ABMFDs are aplastic anemia, myelodysplastic syndrome, and paroxysmal nocturnal hemoglobinuria. Stem cell transplantation is the only treatment that can cure these diseases. However, due to high therapy-related mortality, stem cell transplantation has rarely been used as a first line treatment in treating ABMFD. With the advance of personalized medicine and precision medicine, various novel cellular therapy strategies are in trial to increase the efficiency and efficacy of ABMFD treatment. This article aims to review current available stem cell transplantation protocols and promising cellular therapy research in treating ABMFD.

Updates in the pathophysiological mechanisms of Parkinson's disease: Emerging role of bone marrow mesenchymal stem cells

AIM: To explore the approaches exerted by mesenchymal stem cells(MSCs) to improve Parkinson's disease(PD) pathophysiology.METHODS: MSCs were harvested from bone marrowof femoral bones of male rats, grown and propagated in culture. Twenty four ovariectomized animals were classified into 3 groups: Group(1) was control, Groups(2) and(3) were subcutaneously administered with rotenone for 14 d after one month of ovariectomy for induction of PD. Then, Group(2) was left untreated, while Group(3) was treated with single intravenous dose of bone marrow derived MSCs(BM-MSCs). SRY gene was assessed by PCR in brain tissue of the female rats. Serum transforming growth factor beta-1(TGF-β1), monocyte chemoattractant protein-1(MCP-1) and brain derived neurotrophic factor(BDNF) levels were assayed by ELISA. Brain dopamine DA level was assayed fluorometrically, while brain tyrosine hydroxylase(TH) and nestin gene expression were detected by semi-quantitative real time PCR. Brain survivin expression was determined by immunohistochemical procedure. Histopathological investigation of brain tissues was also done.RESULTS: BM-MSCs were able to home at the injured brains and elicited significant decrease in serum TGF-β1(489.7 ± 13.0 vs 691.2 ± 8.0, P < 0.05) and MCP-1(89.6 ± 2.0 vs 112.1 ± 1.9, P < 0.05) levels associated with significant increase in serum BDNF(3663 ± 17.8 vs 2905 ± 72.9, P < 0.05) and brain DA(874 ± 15.0 vs 599 ± 9.8, P < 0.05) levels as well as brain TH(1.18 ± 0.004 vs 0.54 ± 0.009, P < 0.05) and nestin(1.29 ± 0.005 vs 0.67 ± 0.006, P < 0.05) genes expression levels. In addition to, producing insignificant increase in the number of positive cells for survivin(293.2 ± 15.9 vs 271.5 ± 15.9, P > 0.05) expression. Finally, the brain sections showed intact histological structure of the striatum as a result of treatment with BM-MSCs. CONCLUSION: The current study sheds light on the therapeutic potential of BM-MSCs against PD pathophysiology via multi-mechanistic actions.

Phase 1 human trial of autologous bone marrow-hematopoietic stem cell transplantation in patients with decompensated cirrhosis

AIM: To evaluate safety and feasibility of autologous bone marrow-enriched CD34+ hematopoietic stem cell Tx through the hepatic artery in patients with decompensated cirrhosis.METHODS: Four patients with decompensated cirrhosis were included. Approximately 200 mL of the bone marrow of the patients was aspirated, and CD34+ stem cells were selected. Between 3 to 10 million CD34+ cells were isolated. The cells were slowly infused through the hepatic artery of the patients.RESULTS: Patient 1 showed marginal improvement in serum albumin and no significant changes in other test results. In patient 2 prothrombin time was decreased; however, her total bilirubin, serum creatinine, and Model of End-Stage Liver Disease (MELD) score worsened at the end of follow up. In patient 3 there was improvement in serum albumin, porthrombin time (PT), and MELD score. Patient 4 developed radiocontrast nephropathy after the procedure, and progressed to type 1 hepatorenal syndrome and died of liver failure a few days later. Because of the major side effects seen in the last patient, the trial was prematurely stopped.CONCLUSION: Infusion of CD34+ stem cells through the hepatic artery is not safe in decompensated cirrhosis. Radiocontrast nephropathy and hepatorenal syndrome could be major side effects. However, this study doesnot preclude infusion of CD34+ stem cells through other routes.

Effects of lateral ventricular transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene on cognition in a rat model of Alzheimer's disease

In the present study, transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene into the lateral ventricle of a rat model of Alzheimer＇s disease, resulted in significant attenuation of nerve cell damage in the hippocampal CA1 region. Furthermore, brain-derived neurotrophic factor and tyrosine kinase B mRNA and protein levels were significantly increased, and learning and memory were significantly improved. Results indicate that transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene can significantly improve cognitive function in a rat model of Alzheimer＇s disease, possibly by increasing the levels of brain-derived neurotrophic factor and tyrosine kinase B in the hippocampus.

Autologous bone marrow transplantation in decompensated liver: Systematic review and meta-analysis

AIM:To evaluate the efficacy of autologous bone marrow mononuclear cell transplantation in decompensated liver disease.METHODS:Medline,EMBASE,PubMed,Science Direct,and the Cochrane Library were searched for relevant studies.Retrospective case-control studies were included along with randomized clinical trials.Metaanalysis was performed in line with recommendations from the Cochrane Collaboration software review manager.Heterogeneity was assessed using a randomeffects model.RESULTS:Four randomized controlled trials and four retrospective studies were included.Cell transplantation increased serum albumin level by 1.96 g/L(95%CI:0.74-3.17;P = 0.002],2.55 g/L(95%CI:0.32-4.79;P= 0.03),and 3.65 g/L(95%CI:0.76-6.54;P = 0.01)after 1,3,and 6 mo,respectively.Patients who had undergone cell transplantation also had a lower level of total bilirubin[mean difference(MD):-1.37 mg/dL;95%CI:-2.68-(-0.06);P = 0.04]after 6 mo.This decreased after 1 year when compared to standard treatment(MD:-1.26;95%CI:-2.48-(-0.03);P =0.04].A temporary decrease in alanine transaminase and aspartate transaminase were significant in the cell transplantation group.However,after 6 mo treatment,patients who had undergone cell transplantation had a slightly longer prothrombin time(MD:5.66 s,95%CI:0.04-11.28;P = 0.05).Changes in the model for endstage liver disease score and Child-Pugh score were not statistically significant.CONCLUSION:Autologous bone marrow transplantation showed some benefits in patients with decompensated liver disease.However,further studies are still needed to verify its role in clinical treatment for end-stage liver disease.

Neuronal-like differentiation of bone marrow-derived mesenchymal stem cells induced by striatal extracts from a rat model of Parkinson's disease

A rat model of Parkinson＇s disease was established by 6-hydroxydopamine injection into the medial forebrain bundle. Bone marrow-derived mesenchymal stem cells （BMSCs） were isolated from the femur and tibia, and were co-cultured with 10% and 60% lesioned or intact striatal extracts. The results showed that when exposed to lesioned striatal extracts, BMSCs developed bipolar or multi-polar morphologies, and there was an increase in the percentage of cells that expressed glial fibrillary acidic protein （GFAP）, nestin and neuron-specific enolase （NSE）. Moreover, the percentage of NSE-positive cells increased with increasing concentrations of lesioned striatal extracts. However, intact striatal extracts only increased the percentage of GFAP-positive cells. The findings suggest that striatal extracts from Parkinson＇s disease rats induce BMSCs to differentiate into neuronal-like cells in vitro.

Intracerebroventricular transplanted bone marrow stem cells survive and migrate into the brain of rats with Parkinson's disease

In this study, 6-hydroxydopamine was stereotaxically injected into the right substantia nigra compact and ventral tegmental area of rats to establish Parkinson＇s disease models. The rats then received a transplantation of bone marrow stromal cells that were previously isolated, cultured and labeled with 5-bromo-2＇-deoxyuridine in vitro. Transplantation of the bone marrow stromal cells significantly decreased apomorphine-induced rotation time and the escape latency in the Morris water maze test as compared with rats with untreated Parkinson＇s disease. Immunohistochemical staining showed that, 5-bromo-2＇-deoxyuridine-immunoreactive cells were present in the lateral ventricular wall and the choroid plexus 1 day after transplantation. These immunoreactive cells migrated to the surrounding areas of the lateral cerebral ventricle along the corpus callosum. The results indicated that bone marrow stromal cells could migrate to tissues surround the cerebral ventricle via the cerebrospinal fluid circulation and fuse with cells in the brain, thus altering the phenotype of cells or forming neuron-like cells or astrocytes capable of expressing neuron-specific proteins. Taken together, the present findings indicate that bone marrow stromal cells transplanted intracerebroventricularly could survive, migrate and significantly improve the rotational behavior and cognitive function of rats with experimentally induced Parkinson＇s disease.

Repeated application of autologous bone marrow-derived stem cell therapy in patients with severe Buerger’s disease

Stem cell therapy (SCT) is a promising and prospective approach in the treatment of patients with severe peripheral arterial disorders, primarily with Buerger’s disease. However, very little is known about the duration of the effect of SCT, and to our best knowledge no data are available on the efficacy and safety of repeated SCT in patients with Buerger’s disease. Here we report on two patients with severe Buerger’s disease, who received repeated autologous bone marrow-derived stem cell therapy. Our results show that a second SCT, applied to a previously treated leg 30 or 36 months after the first treatment was efficient in both cases. After twelve months, the clinical state of the repeatedly treated lower limb improved spectacularly and non-healing ulcers healed more rapidly than after the first SCT. No severe adverse events were detected. Thus repeated SCT offers a safe and efficient treatment option for relapsing patients at the advanced stage of Buerger’s disease.

Protective effect of bone marrow-derived mesenchymal stem cells on dopaminergic neurons against 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in rat brain slices

BACKGROUND： To date, the use of bone marrow-derived mesenchymal stem cells （MSCs） for the treatment of Parkinson’s disease have solely focused on in vivo animal models. Because of the number of influencing factors, it has been difficult to determine a consistent outcome. OBJECTIVE： To establish an injury model in brain slices of substantia nigra and striatum using 1-methyl-4-phenylpytidinium ion （MPP＋）, and to investigate the effect of MSCs on dopaminergic neurons following MPP＋ induced damage. DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, animal experiment using I mmunohistochemistry was performed at the Laboratory of the Department of Anatomy, Fudan University between January 2004 and December 2006. MATERIALS： Primary MSC cultures were obtained from femurs and tibias of adult Sprague Dawley rats. Organotypic brain slices were isolated from substantia nigra and striatum of 1-day-old Sprague Dawley rat pups. Monoclonal antibodies for tyrosine hydroxylase （TH, 1：5 000） were from Santa Cruz （USA）; goat anti-rabbit IgG antibodies labeled with FITC were from Boster Company （China）. METHODS： Organotypic brain slices were cultured for 5 days in whole culture medium supplemented with 50% DMEM, 25% equine serum, and 25% Tyrode’s balanced salt solution. The medium was supplemented with 5 μg/mL Ara-C, and the culture was continued for an additional 5 days. The undergrowth of brain slices was discarded at day 10. Eugonic brain slices were cultured with basal media for an additional 7 days. The brain slices were divided into three groups： control, MPP＋ exposure, and co-culture. For the MPP＋ group, MPP＋ （30 μmol/L） was added to the media at day 17 and brain slices were cultured for 4 days, followed by control media. For the co-culture group, the MPP＋ injured brain slices were placed over MSCs in the well and were further cultured for 7 days. MAIN OUTCOME MEASURES： After 28 days in culture, neurite outgrowth was examined in the brain slices under phase-contrast microscopy. The percent of area containing dead cells in each brain slice was calculated with the help of propidium iodide fluorescence. Brain slices were stained with antibodies for TH to indicate the presence of dopaminergic neurons. Transmission electron microscopy was applied to determine the effect of MSCs on neuronal ultrastructure. RESULTS： Massive cell death and neurite breakage was observed in the MPP＋ group. In addition, TH expression was significantly reduced, compared to the control group （P 〈 0.01）. After 7 days in culture with MSCs, the co-culture group presented with less cell damage and reduced neurite breakage, and TH expression was increased. However, these changes were not significantly different from the MPP＋ group （P 〈 0.01）. Electron microscopy revealed reduced ultrastructural injury to cells in the brain slices. However, vacuoles were present in cells, with some autophagic vacuoles. CONCLUSION： Bone marrow-derived MSCs can promote survival of dopaminergic neurons following MPP＋-induced neurotoxicity in co-cultures with substantia nigra and striatum brain slices.

Thiopurine S-methyltransferase polymorphisms and thiopurine toxicity in treatment of inflammatory bowel disease

AIM: To evaluate the relationship between thiopu- rine S-methyltransferase (TPMT) polymorphisms and thiopurine-induced adverse drug reactions (ADRs) in inflammatory bowel disease (IBD). METHODS: Eligible articles that compared the frequency of TPMT polymorphisms among thiopurine-tolerant and-intolerant adult IBD patients were included. Statistical analysis was performed with Review Manager 5.0. Sub-analysis/sensitivity analysis was also performed. RESULTS: Nine studies that investigated a total of 1309 participants met our inclusion criteria. The inci- dence of TPMT gene mutation was increased 2.93-fold (95% CI: 1.68-5.09, P = 0.0001) and 5.93-fold (95% CI: 2.96-11.88, P < 0.00001), respectively, in IBD patients with thiopurine-induced overall ADRs and bone marrow toxicity (BMT), compared with controls. The OR for TPMT gene mutation in IBD patients withthiopurine-induced hepatotoxicity and pancreatitis was 1.51 (95% CI: 0.54-4.19, P = 0.43) and 1.02 (95% CI: 0.26-3.99, P = 0.98) vs controls, respectively. CONCLUSION: This meta-analysis suggests that the TPMT polymorphisms are associated with thiopurine-induced overall ADRs and BMT, but not with hepatotoxicity and pancreatitis.

Stem Cell Ophthalmology Treatment Study (SCOTS)： bone marrow-derived stem cells in the treatment of Leber＇s hereditary optic neuropathy

The Stem Cell Ophthalmology Treatment Study （SCOTS） is currently the largest-scale stem cell ophthal- mology trial registered at ClinicalTrials.gov （identifier： NCT01920867）. SCOTS utilizes autologous bone marrow-derived stem cells （BMSCs） to treat optic nerve and retinal diseases. Treatment approaches include a combination of retrobulbar, subtenon, intravitreal, intra-optic nerve, subretinal, and intravenous injection of autologous BMSCs according to the nature of the disease, the degree of visual loss, and any risk factors related to the treatments. Patients with Leber＇s hereditary optic neuropathy had visual acuity gains on the Early Treatment Diabetic Retinopathy Study （ETDRS） of up to 35 letters and Snellen acuity improvements from hand motion to 20/200 and from counting fingers to 20/100. Visual field improvements were noted. Macular and optic nerve head nerve fiber layer typically thickened. No serious complications were seen. The increases in visual acuity obtained in our study were encouraging and suggest that the use of autolo- gous BMSCs as provided in SCOTS for ophthalmologic mitochondrial diseases including Leber＇s hereditary optic neuropathy may be a viable treatment option.

Hematopoietic cell transplantation for Crohn’s disease;is it time?

AIM: To review all studies in the literature that have assessed Hematopoietic cell transplantation (HCT) and Crohn’s disease (CD) with the ultimate aims of determining if this is a viable treatment option for those with CD. A secondary aim was to review the above literature and determine if the studies shed further light on the mechanisms involved in the pathogenesis of CD. METHODS: An extensive Medline search was performed on all articles from 1970 to 2005 using the ; bone marrow transplant, stem cell, hematopoietic cell, Crohn’s disease and inflammatory bowel disease. RESULTS: We identified one case in which a patient developed CD following an allogeneic HCT from a sibling suffering with CD. Evidence for transfer of the genetic predisposition to develop CD was also identified with report of a patient that developed severe CD following an allogeneic HCT. Following HCT it was found that the donor (that had no signs or symptoms of CD) and the recipient had several haplotype mismatches in HLA class Ⅲ genes in the IBD3 locus including a polymorphism of NOD2/CARD15 that has been associated with CD. Thirty three published cases of patients with CD who underwent either autologous or allogeneic HCT were identified. At the time of publication 29 of these 33 patients were considered to be in remission. The median follow-up time was seven years, and twenty months for allogeneic and autologous HCT respectively.For patients who underwent HCT primarily for treatment of their CD there have been no mortalities related to transplant complications. CONCLUSION: Overall these preliminary data suggest that both allogeneic and autologous HCT may be effective in inducing remission in refractory CD. Thissupports the hypothesis that the hematolymphatic cells play a key role in CD and that resetting of the immune system may be a critical approach in the management or cure of CD.

Bone marrow-derived mesenchymal stem cells increase dopamine synthesis in the injured striatum

Previous studies showed that tyrosine hydroxylase or neurturin gene-modified cells transplanted into rats with Parkinson＇s disease significantly improved behavior and increased striatal dopamine content. In the present study, we transplanted tyrosine hydroxylase and neurturin gene-modified bone marrow-derived mesenchymal stem cells into the damaged striatum of Parkinson＇s disease model rats. Several weeks after cell transplantation, in addition to an improvement of motor function tyrosine hydroxylase and neurturin proteins were up-regulated in the injured striatum, and importantly, levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid increased significantly. Furthermore, the density of the D2 dopamine receptor in the postsynaptic membranes of dopaminergic neurons was decreased. These results indicate that transplantation of tyrosine hydroxylase and neurturin gene-modified bone marrow-derived mesenchymal stem cells increases dopamine synthesis and significantly improves the behavior of rats with Parkinson＇s disease.

Reduced glutathione alleviates the toxic effect of 6-hydroxydopamine on bone marrow stromal cells

We studied the effect of reduced glutathione on bone marrow stromal cells （BMSCs） treated with 6-hydroxydopamine （6-OHDA）, which shows a toxic effect on dopaminergic neurons. The proliferation of BMSCs treated with 6-OHDA decreased, while that of BMSCs treated with reduced glutathione increased. The proliferation of BMSCs treated with both 6-OHDA and reduced glutathione was significantly higher compared with that treated with 6-OHDA alone. These findings indicate that reduced glutathione alleviates the toxic effect of 6-OHDA on BMSCs.

Influence of partially purified lymphocyte chalone on bone marrow transplantation in mice

Lymphocyte chalone was partially purified by ultrafiltration and chromatography onSep hadex G75 column.Its inhibitory effect on proliferation of T,B lymphoctes was demonstratedby[~3H]-TdR incorporation method.In mouse bone marrow transplantation model,the treatmentwith lymphocyte chalone in vivo and in vitro was found to have significantly improved the 30 daysurvival rate and mean survival time,suggesting its inhibitory effect on acute graft-versus-host dis-ease(GVHD)and the helpful effect on H-2 incompatible tone marrow transplantation in mice.

Attenuation of GVHD for Allo-Bone Marrow Transplantation Recipient by FasL-Fas Pathway in an H-2 Haplotype Disparate Mouse Combination

In order to explore a new special and effective way to prevent graft versus host disease (GVHD) after allogenic bone marrow transplantation (allo-BMT), the stem cell antigen-1 (Sca-1) + early hematopoietic cells (EHC) from BALB/c mouse (H-2 d) were introduced with exogenous mouse Fas ligand (mFasL) cDNA gene by the retrovirus-mediated gene transfer and expanded for one week, and then they were co-cultured with the spleen mononuclear cells (SMNC) from BAC mouse (H-2 d×b) as one way mixed lymphocyte reaction (OWMLR). The cytotoxicity of treated BAC mouse SMNC against Na 2 51CrO 4 labeling SMNC from BALB/c mouse was observed. The bone marrow mononuclear cells (BMMNC) from BAC mouse treated by the above methods were transplanted into lethally-irradiated congenic BALB/c mice to observe the occurrence of GVHD. The results showed that the SMNC from BAC mouse after OWMLR with exogenous mFasL cDNA gene-transduced hematopoietic cells (HC) from BALB/c mouse in a ratio of 1 to 5 exhibited an obvious inhibition of the cytotoxicity against the BALB/c mouse spleen cells at different effector/target ratios as compared to the control group (P<0.01). The gradeⅠ GVHD or no GVHD and the 80 % survival rate at day 60 post-BMT were observed in the BALB/c mouse receiving BAC mouse BMMNC treated with similar way, while the grade Ⅱ-Ⅲ GVHD and the 20 % survival rate were noted in the control group (P<0.01). It is suggested that the attenuation of GVHD in allo-BMT recipient could be successfully achieved through FasL-Fas pathway in an H-2 haplotype disparate mouse combination.

Acute GvHD and the cutaneous ultrastructural changes in mismatched bone marrow transplantation

Six patients treated with human leukocyte antigen (HLA)-mismatched bone marrow transplantation (BMT) suffered from grade I to IV acute graft-versus-host disease (aGvHD) after engrafting. Up to date, 4 patients with grade I to II GvHD have lived for over 2920, 910, 740 and 680 days, respectively. Two other patients died of grade IV hyperacute GvHD. The results seem to indicate that patients in mismatched BMT have a high incidence of aGvHD within a month. The severity of aGvHD is positively correlated with the degree of HLA mismatching. The higher the degree of mismatch of HLA, the earlier and the more severe the aGvHD occurrs. The cutaneous lesion of the patient with GvHD is severe and of ten complicated by mucositis. Lethal hyperacute GvHD must be considered when a patient shows following signs at beginning: (1) The symptoms appear early (within 2weeks) ;(2) peripheral white blood cell count does not recover (<0. 5×109/L) to normal; and (3) high fever persists. In the epidermal ultrastructure of patients, besides acantholysis, autophagic degeneration of keratinocytes,and satellite cell dyskeratosis, there were scattered necrotic keratinocytes, breaking and thickening of basal membrane and presence of a lot of pigment in the intercellular space. These imply that the ultrastructural damages in the skin of patients with aGvHD after mismatched transplantation are more severe than after matched ones.

Effect of NK cells on GVHD in H-2 haploidentical bone marrow transplantation in mice

To study the effect of natural killer （NK） cells on graft-versus-host disease （GVHD） after H-2 haploidentical bone marrow transplantation （BMT） in mice. Methods ：Murine model of H-2 haploidentical BMT was established by using Balb/c （H- 2d） mouse as recipient, and Balb/c （H-2d）×C57BL/6 （H-2b） （H-2db） mouse as donor. Lethally irradiated Balb/c （H-2d） mice were transplanted with the bone marrow cells from Balb/c（H-2d）×C57BL/6（H-2b） （H-2db） mice containing donor spleen cells and/or NK cells. GVHD and survival rates were studied by observation of clinical manifestations and pathological changes. Results：In the group of bone marrow ＋spleen cells, GVHD was induced in 90% mice; but in the group plus with low amount of NK cells, GVHD was induced in 20% mice; and in the group transplanted with high amount of NK cells, GVHD was induced only in 10% mice. Compared to the group transplanted only with BM plus spleen cells, the incidences of GVHD in the latter two groups decreased significantly （P 〈 0.01） and the survival rates at different periods of 15, 30, 45 and 60 days increased obviously （P 〈 0.01）. Conclusion： In mouse H-2 haploidentical BMT, alloreactive NK cells can reduce the incidence of GVHD and increase the survival rate.