Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

Feasibility of autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold for cartilage tissue engineering

Extracellular matrix (ECM) materials are widely used in cartilage tissue engineering. However, the current ECM materials are unsatisfactory for clinical practice as most of them are derived from allogenous or xenogenous tissue. This study was designed to develop a novel autologous ECM scaffold for cartilage tissue engineering. The autologous bone marrow mesenchymal stem cell-derived ECM (aBMSC-dECM) membrane was collected and fabricated into a three-dimensional porous scaffold via cross-linking and freeze-drying techniques. Articular chondrocytes were seeded into the aBMSC-dECM scaffold and atelocollagen scaffold, respectively. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The current results showed that the aBMSC-dECM scaffold had a good microstructure and biocompatibility. After 4 weeks in vitro culture, the engineered cartilage in the aBMSC-dECM scaffold group formed thicker cartilage tissue with more homogeneous structure and higher expressions of cartilaginous gene and protein compared with the atelocollagen scaffold group. Furthermore, the engineered cartilage based on the aBMSC-dECM scaffold showed better cartilage formation in terms of volume and homogeneity, cartilage matrix content, and compressive modulus after 3 weeks in vivo implantation. These results indicated that the aBMSC-dECM scaffold could be a successful novel candidate scaffold for cartilage tissue engineering.

Selective enhancement of donor hematopoietic cell engraftment by the CXCR4 antagonist AMD3100 in a mouse transplantation model.

The interaction between stromal cell-derived factor-1 (SDF-1) with CXCR4 chemokine receptors plays an important role in hematopoiesis following hematopoietic stem cell transplantation. We examined the efficacy of post transplant administration of a specific CXCR4 antagonist (AMD3100) in improving animal survival and in enhancing donor hematopoietic cell engraftment using a congeneic mouse transplantation model. AMD3100 was administered subcutaneously at 5 mg/kg body weight 3 times a week beginning at day +2 post-transplant. Post-transplant administration of AMD3100 significantly improves animal survival. AMD3100 reduces pro-inflammatory cytokine/chemokine production. Furthermore, post transplant administration of AMD3100 selectively enhances donor cell engraftment and promotes recovery of all donor cell lineages (myeloid cells, T and B lymphocytes, erythrocytes and platelets). This enhancement results from a combined effect of increased marrow niche availability and greater cell division induced by AMD3100. Our studies shed new lights into the biological roles of SDF-1/CXCR4 interaction in hematopoietic stem cell engraftment following transplantation and in transplant-related mortality. Our results indicate that AMD3100 provides a novel approach for enhancing hematological recovery following transplantation, and will likely benefit patients undergoing transplantation.

Efficacy of Pharmacokinetics-Directed Busulfan, Cyclophosphamide, and Etoposide Conditioning and Autologous Stem Cell Transplantation for Lymphoma: Comparison of a Multicenter Phase II Study and CIBMTR Outcomes.

Busulfan, cyclophosphamide, and etoposide (BuCyE) is a commonly used conditioning regimen for autologous stem cell transplantation (ASCT). This multicenter, phase II study examined the safety and efficacy of BuCyE with individually adjusted busulfan based on preconditioning pharmacokinetics. The study initially enrolled Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) patients ages 18 to 80 years but was amended due to high early treatment-related mortality (TRM) in patients > 65 years. BuCyE outcomes were compared with contemporaneous recipients of carmustine, etoposide, cytarabine, and melphalan (BEAM) from the Center for International Blood and Marrow Transplant Research. Two hundred seven subjects with HL (n = 66) or NHL (n = 141) were enrolled from 32 centers in North America, and 203 underwent ASCT. Day 100 TRM for all subjects (n = 203), patients > 65 years (n = 17), and patients ≤ 65 years (n = 186) were 4.5%, 23.5%, and 2.7%, respectively. The estimated rates of 2-year progression-free survival (PFS) were 33% for HL and 58%, 77%, and 43% for diffuse large B cell lymphoma (DLBCL; n = 63), mantle cell lymphoma (MCL; n = 29), and follicular lymphoma (FL; n = 23), respectively. The estimated rates of 2-year overall survival (OS) were 76% for HL and 65%, 89%, and 89% for DLBCL, MCL, and FL, respectively. In the matched analysis rates of 2-year TRM were 3.3% for BuCyE and 3.9% for BEAM, and there were no differences in outcomes for NHL. Patients with HL had lower rates of 2-year PFS with BuCyE, 33% (95% CI, 21% to 46%), than with BEAM, 59% (95% CI, 52% to 66%), with no differences in TRM or OS. BuCyE provided adequate disease control and safety in B cell NHL patients ≤ 65 years but produced worse PFS in HL patients when compared with BEAM.

Bioreactor Expansion of Human Adult Bone Marrow-Derived Mesenchymal Stem Cells

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell transplantation (HSCT) alleviates complications such as graft-versus-host disease, leading to a speedy recovery of hematopoiesis. To meet such clinical demand, a fast MSCs expansion method is required. In the present study, we examined the feasibility of expanding MSCs from the isolated bone marrow mononuclear cells using a rotary bioreactor system. The cells were cultured in a rotary bioreactor with Myelocult� medium containing a combination of supplementary factors, including stem cell factor (SCF), interleukin 3 and 6 (IL-3, IL-6). After 8 days of culture, total cell numbers, Stro-1+CD44+CD34- MSCs and CD34+CD44+Stro-1- HSCs were increased 9, 29, and 8 folds respectively. Colony forming efficiency-fibroblast per day (CFE-F/day) of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSCs markers endoglin (SH2) and vimentin, whereas markers associated with lineage differentiation including osteocalcin (osteogenesis), Type II collagen (chondrogenesis) and C/EBPα (adipogenesis) were not detected. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocytes and adipocytes. Taken together, we conclude that the rotary bioreactor with the modified Myelocult� medium reported in this study may be used to rapidly expand MSCs.

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

Hematopoietic stem cell transplantation in Switzerland: a comprehensive quality control report on centre effect.

Interest groups advocate centre-specific outcome data as a useful tool for patients in choosing a hospital for their treatment and for decision-making by politicians and the insurance industry. Haematopoietic stem cell transplantation (HSCT) requires significant infrastructure and represents a cost-intensive procedure. It therefore qualifies as a prime target for such a policy. We made use of the comprehensive database of the Swiss Blood Stem Cells Transplant Group (SBST) to evaluate potential use of mortality rates. Nine institutions reported a total of 4717 HSCT - 1427 allogeneic (30.3%), 3290 autologous (69.7%) - in 3808 patients between the years 1997 and 2008. Data were analysed for survival- and transplantation-related mortality (TRM) at day 100 and at 5 years. The data showed marked and significant differences between centres in unadjusted analyses. These differences were absent or marginal when the results were adjusted for disease, year of transplant and the EBMT risk score (a score incorporating patient age, disease stage, time interval between diagnosis and transplantation, and, for allogeneic transplants, donor type and donor-recipient gender combination) in a multivariable analysis. These data indicate comparable quality among centres in Switzerland. They show that comparison of crude centre-specific outcome data without adjustment for the patient mix may be misleading. Mandatory data collection and systematic review of all cases within a comprehensive quality management system might, in contrast, serve as a model to ascertain the quality of other cost-intensive therapies in Switzerland.

Impact de la maladie du greffon contre l’hôte sur la reconstitution immunitaire suite à une transplantation allogénique de cellules souches hématopoïétiques

La transplantation allogénique de cellules souches hématopoïétiques (ASCT) est couramment utilisée pour traiter différents cancers hématologiques. Malheureusement, l’effet bénéfique de cette technique est limité par la réaction du greffon contre l’hôte (GVHD) qui demeure la cause principale de mortalité post-greffe. La GVHD endommage différents organes et retarde la reconstitution immunitaire des lymphocytes T (LT) ce qui augmente les risques d’infection et de rechute. Le développement de nouveaux traitements permettant d’accélérer la reconstitution immunitaire augmenterait donc les chances de survie des patients greffés. Il existe deux façons de régénérer des LT: via la thymopoïèse qui consiste à produire de nouveaux LT, ou par la prolifération homéostatique (PH) qui implique l’expansion rapide des LT matures retrouvés dans le greffon. La PH requiert deux signaux essentiels: l’interleukine-7 (IL-7) et la présentation d’antigènes du soi par les cellules dendritiques (DC) via le complexe majeur d’histocompatibilité (CMH) I pour les LT CD8+ et le CMH II pour les LT CD4+. Dans un contexte d’ASCT, la chimiothérapie et la GVHD endommagent le thymus rendant la thymopoïèse inefficace. Par conséquent, la reconstitution immunitaire repose presque entièrement sur la PH des LT. L’objectif de cette thèse était de comprendre comment la GVHD affecte la reconstitution des LT. Grâce à un modèle murin, nous avons démontré que la PH des LT CD4+ est absente durant la GVHD et ce, dû à de faibles niveaux d’IL-7 et une diminution du nombre de DC. La perte des DC est en grande partie causée par des niveaux réduits de stromal derived factor-1α (SDF-1α) et par l’absence de progéniteurs de DC dans la moelle osseuse des souris en GVHD. Le traitement des souris en GVHD avec du SDF-1α permet d’augmenter le nombre de DC, et lorsqu’administré avec l’IL-7, améliore significativement la PH des LT CD4+. Contrairement aux LT CD4+, l’administration d’IL-7 seule est suffisante pour restaurer la PH des LT CD8+ durant la GVHD et ce, même en absence des DC. Ces différences s’expliquent pour deux raisons : 1) l’expression du CMH I, contrairement au CMH II, n’est pas limitée aux DC mais est également exprimée par les cellules stromales du receveur ce qui est suffisant pour induire la PH des LT CD8+ et 2) les LT CD8+ répondent à des concentrations plus faibles d’IL-7 systémique comparativement aux LT CD4+. En conclusion, l’ensemble de ces résultats permettra de mettre en place des études translationnelles sur le potentiel thérapeutique du SDF-1α et de l’IL-7 dans la reconstitution immunitaire des patients greffés.

Expression of Pancreatic Endocrine Markers by Prolactin-Treated Rat Bone Marrow Mesenchymal Stem Cells

Background. Mesenchymal stem cells (MSCs) are an attractive source for generation of cells with beta-cell properties. Previous studies have demonstrated the ability of prolactin to induce an increase in beta-cell mass and maturation, which suggests beneficial effects of its use in MSC differentiation protocols. Objective. To evaluate the expression of endocrine differentiation markers in rat MSCs treated in vitro with prolactin. Methods. Mesenchymal stem cells from bone marrow of Wistar rats were isolated, expanded, and characterized. Differentiation of MSCs was induced in medium containing 23 mmol/L of glucose, and nicotinamide, 2-mercaptoethanol, and exendin-4, in the presence or absence of 500 ng/mL of rat recombinant prolactin. Expression of endocrine markers and prolactin receptor genes was evaluated using real-time polymerase chain reaction, and compared between culture stages and presence vs absence of prolactin in the culture medium. Expression of insulin, somatostatin, glucagon, and pancreatic and duodenal homeobox 1 was also evaluated at immunofluorescence microscopy. Results. Isolated cells were mostly MSCs, as confirmed at fluorescent-activated cell sorting and cytochemistry. Pax6, Ngn-3, Isl1, NeuroD1, Nkx2.2, and Nkx6.1 exhibited varied expression during culture stages. The long form of the prolactin receptor messenger RNA was induced in prolactin-treated cultures (P < .05). The somatostatin gene was induced in early stages of differentiation (P < .05), and its expression was induced by prolactin, as confirmed using immunofluorescence. Conclusion. Culture of rat bone marrow MSCs in differentiation medium induces expression of pancreatic endocrine-specific genes, and somatostatin and prolactin receptor expression was also induced by prolactin.

Cardiac function in dogs with chronic Chagas cardiomyopathy undergoing autologous stem cell transplantation into the coronary arteries

This study assessed the effects of a single intracoronary injection of autologous stem cells on the cardiac function of dogs with Chagas cardiomyopathy. Bone-marrow-derived stem cells were delivered into the right and left coronary arteries of 5 mature dogs with mildly compromised cardiac function due to chronic Chagas cardiomyopathy. Blood pressure and electrocardiographic and echocardiographic parameters were recorded at monthly intervals for 6 mo in the 3 dogs that survived. Although no changes were observed in the electrocardiogram and blood pressure, there was a significant increase in peak velocity of aortic flow 3 mo after stem cell transplantation. Pre-ejection period, isovolumic relaxation time, and the Tei index of myocardial performance were reduced significantly 4 mo after the procedure. All significant changes persisted to the end of the study. The results suggest that the transplantation of autologous bone-marrow-derived stem cells into the coronary arteries of dogs with Chagas cardiomyopathy may have a beneficial effect but the small number of dogs studied was a limitation.

Graft versus host disease in a rat small bowel transplant model after T-cell depleted donor specific bone marrow infusion

Baixas doses de irradiação associadas à infusão de células da medula óssea não previnem a ocorrência da reação do enxerto versus hospedeiro após o transplante intestinal. OBJETIVO: Neste estudo foi avaliado a potencial vantagem em estender o regime imunossupressor associado a infusão de células de medula óssea do doador depletadas de células T na prevenção da reação do enxerto versus hospedeiro após o transplante intestinal. MÉTODOS: Transplante heterotópico de intestino delgado foi realizado em ratos Lewis como receptores e da como doadores, distribuídos em cinco grupos de acordo com a duração da imunossupressão, irradiação e do uso de medula óssea normal ou depletada: G1 (n=6), sem irradiação e G2 (n=9), G3 (n=4), G4 (n=5) e G5 (n=6) foram irradiados com 250 rd. Grupos1, 2, 4 e G3 e 5 foram infundidos com 100 x 10(6) células da medula normal e depletada respectivamente. Animais no G1,2,3 foram imunossuprimidos com 1mg/kg/FK506/ IM por cinco dias e G4 e cinco por 15 dias. Anticorpos monoclonais contra células CD3 e colunas magnéticas foram utilizadas para a depleção da medula óssea. Os animais foram examinados para a presença de rejeição, reação do enxerto versus hospedeiro, chimerismo e biópsias intestinais e da pele. RESULTADOS: Rejeição mínima foi observada em todos os grupos; entretanto, a reação do enxerto versus hospedeiro somente nos animais irradiados. Extensão da imunossupressão alterou a gravidade da reação nos animais dos G4 e 5. Rejeição foi a causa mortis no G1 e a reação do enxerto versus hospedeiro nos Grupos 2,3,4 e 5, não controlada com a infusão de medula óssea depletada. O chimerismo total e de células T do doador foi estatisticamente maior nos grupos irradiados em comparação ao G1. CONCLUSÃO: A extensão do regime de imunossupressão associado a baixas doses de irradiação diminui a gravidade da reação do enxerto versus hospedeiro, não abolida pelo uso de medula óssea depletada.

Bone marrow cells of swine: collection and separation

Bone marrow is a source of stem cells for greater and easier access, which is widely studied as a provider of hematopoietic and mesenchymal cells for various purposes, mainly therapeutic by the advances in research involving cell therapy. The swine is an animal species commonly used in the pursuit of development of experimental models. Thus, this study aimed to standardize protocol for collection and separation of bone marrow in swines, since this species is widely used as experimental models for various diseases. Twelve animals were used, which underwent bone marrow puncture with access from the iliac crest and cell separation by density gradient followed by a viability test with an average of 98% of viable cells. Given our results, we can ensure the swine as an excellent model for obtaining and isolation of mononuclear cells from bone marrow, stimulating several studies addressing the field of cell therapy. Microsc. Res. Tech., 2012. (C) 2012 Wiley Periodicals, Inc.

Protective effects of bone marrow mononuclear cell therapy on lung and heart in an elastase-induced emphysema model

We hypothesized that bone marrow-derived mononuclear cell (BMDMC) therapy protects the lung and consequently the heart in experimental elastase-induced emphysema. Twenty-four female C57BL/6 mice were intratracheally instilled with saline (C group) or porcine pancreatic elastase (E group) once a week during 4 weeks. C and E groups were randomized into subgroups receiving saline (SAL) or male BMDMCs (2 x 10(6), CELL) intravenously 3 h after the first saline or elastase instillation. Compared to E-SAL group, E-CELL mice showed, at 5 weeks: lower mean linear intercept, neutrophil infiltration, elastolysis, collagen fiber deposition in alveolar septa and pulmonary vessel wall, lung cell apoptosis, right ventricle wall thickness and area, higher endothelial growth factor and insulin-like growth factor mRNA expressions in lung tissue, and reduced platelet-derived growth factor, transforming growth factor-beta, and caspase-3 expressions. In conclusion, BMDMC therapy was effective at modulating the inflammatory and remodeling processes in the present model of elastase-induced emphysema. (c) 2012 Elsevier B.V. All rights reserved.

Healthcare-associated infection in hematopoietic stem cell transplantation patients: risk factors and impact on outcome

Objective: The objective of this study was to analyze the incidence of and risk factors for healthcare-associated infections (HAI) among hematopoietic stem cell transplantation (HSCT) patients, and the impact of such infections on mortality during hospitalization. Methods: We conducted a 9-year (2001-2009) retrospective cohort study including patients submitted to HSCT at a reference center in Sao Paulo, Brazil. The incidence of HAI was calculated using days of neutropenia as the denominator. Data were analyzed using EpiInfo 3.5.1. Results: Over the 9-year period there were 429 neutropenic HSCT patients, with a total of 6816 days of neutropenia. Bloodstream infections (BSI) were the most frequent infection, presenting in 80 (18.6%) patients, with an incidence of 11.7 per 1000 days of neutropenia. Most bacteremia was due to Gram-negative bacteria: 43 (53.8%) cases were caused by Gram-negative species, while 33 (41.2%) were caused by Gram-positive species, and four (5%) by fungal species. Independent risk factors associated with HAI were prolonged neutropenia (odds ratio (OR) 1.07, 95% confidence interval (CI) 1.04-1.10) and duration of fever (OR 1.20, 95% CI 1.12-1.30). Risk factors associated with death in multivariate analyses were age (OR 1.02, 95% CI 1.01-1.43), being submitted to an allogeneic transplant (OR 3.08, 95% CI 1.68-5.56), a microbiologically documented infection (OR 2.96, 95% CI 1.87-4.6), invasive aspergillosis disease (OR 2.21, 95% CI 1.1-4.3), and acute leukemias (OR 2.24, 95% CI 1.3-3.6). Conclusions: BSI was the most frequent HAI, and there was a predominance of Gram-negative microorganisms. Independent risk factors associated with HAI were duration of neutropenia and fever, and the risk factors for a poor outcome were older age, type of transplant (allogeneic), the presence of a microbiologically documented infection, invasive aspergillosis, and acute leukemia. Further prospective studies with larger numbers of patients may confirm the role of these risk factors for a poor clinical outcome and death in this transplant population. (C) 2012 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.