Sustained improvement in left ventricular function after bone marrow derived cell therapy in patients with acute ST elevation myocardial infarction. A 5-year follow-up from the Stem Cell Transplantation in Ischaemic Myocardium Study.

BACKGROUND: Intracoronary injection of autologous bone marrow-derived mononucleated cells (BM-MNC) may improve LV function shortly after acute ST elevation myocardial infarction (STEMI), but little is known about the long-term durability of the treatment effect. METHODS: In a single-centre trial a total of 60 patients with acute anterior STEMI, successful reperfusion therapy and a left ventricular ejection fraction (LVEF) of <50% were screened for the study. 23 patients were actively treated with intracoronary infusion of BM-MNC within a median of 3 days. The open-label control group consisted of 19 patients who did not consent to undergo BM-MNC treatment but agreed to undergo regular clinical and echocardiographic follow-up for up to 5 years after AMI. RESULTS: Whereas at 4 months there was no significant difference between the increase in LVEF in the BM-MNC group and the control group (+7.0%, 95%CI 3.6; 10.4) vs. +3.9%, 95%CI -2.1; 10), the absolute increase at 5 years remained stable in the BM-MNC but not in the control group (+7.95%, 95%CI 3.5; 12.4 vs. -0.5%, 95%CI -5.4; 4.4; p for interaction between groups = 0.035). DISCUSSION: In this single-centre, open-labelled study, intracoronary administration of BM-MNC is feasible and safe in the short term. It is also associated with sustained improvement of left ventricular function in patients with acute myocardial infarction, encouraging phase III studies to examine the potential BM-MNC effect on clinical outcome.

Value of allogeneic versus autologous stem cell transplantation and chemotherapy in patients with myelodysplastic syndromes and secondary acute myeloid leukemia. Final results of a prospective randomized European Intergroup Trial.

BACKGROUND: Allogeneic stem cell transplantation is usually considered the only curative treatment option for patients with advanced or transformed myelodysplastic syndromes in complete remission, but post-remission chemotherapy and autologous stem cell transplantation are potential alternatives, especially in patients over 45 years old. DESIGN AND METHODS: We evaluated, after intensive anti-leukemic remission-induction chemotherapy, the impact of the availability of an HLA-identical sibling donor on an intention-to treat basis. Additionally, all patients without a sibling donor in complete remission after the first consolidation course were randomized to either autologous peripheral blood stem cell transplantation or a second consolidation course consisting of high-dose cytarabine. RESULTS: The 4-year survival of the 341 evaluable patients was 28%. After achieving complete remission, the 4-year survival rates of patients under 55 years old with or without a donor were 54% and 41%, respectively, with an adjusted hazard ratio of 0.81 (95% confidence interval [95% CI], 0.49-1.35) for survival and of 0.67 (95% CI, 0.42-1.06) for disease-free survival. In patients with intermediate/high risk cytogenetic abnormalities the hazard ratio in multivariate analysis was 0.58 (99% CI, 0.22-1.50) (P=0.14) for survival and 0.46 (99% CI, 0.22-1.50) for disease-free survival (P=0.03). In contrast, in patients with low risk cytogenetic characteristics the hazard ratio for survival was 1.17 (99% CI, 0.40-3.42) and that for disease-free survival was 1.02 (99% CI, 0.40-2.56). The 4-year survival of the 65 patients randomized to autologous peripheral blood stem cell transplantation or a second consolidation course of high-dose cytarabine was 37% and 27%, respectively. The hazard ratio in multivariate analysis was 1.22 (95% CI, 0.65-2.27) for survival and 1.02 (95% CI, 0.56-1.85) for disease-free survival. CONCLUSIONS: Patients with a donor and candidates for allogeneic stem cell transplantation in first complete remission may have a better disease-free survival than those without a donor in case of myelodysplastic syndromes with intermediate/high-risk cytogenetics. Autologous peripheral blood stem cell transplantation does not provide longer survival than intensive chemotherapy.

Cell Adhesion Peptide RGDSP and Laminin Support Proliferation and Migration of Postnatal Retinal Stem Cells in a 3D Hydrogel Culture System

Purpose: Retinal stem cells (RSCs) can be isolated from radial glia population of the newborn mouse retina (Angénieux et al., 2006). These RSCs have great capacity to renew and generate neurons including cells differentiated towards the photoreceptor lineage (Mehri-Soussi et al., 2006). However, our published results showed poor integration and survival rate after cell grafting into the retina. The uncontrollable environment of retina seems to be the problem. To bypass this, we are trying to generate hemi-retinal tissue in vitro that can be used for transplantation. Methods: Expanded RSCs were seeded in a mixture of poly-ethylene-glycol (PEG)-polymer-based hydrogels crosslinked by peptides that also serve as substrates for matrix metalloproteinases. Different doses of crosslinker peptides were tested. Several growth factors were studied to stimulate cell proliferation and differentiation. Results: Cells were trapped in hydrogels and cultured in the presence of FGF2 and EGF. Spherical cell clusters indicating proliferation appeared within several days, but there was no cell migration within the gel. We then added cell adhesion molecules integrin ligand RGDSP, or laminin, or a combination of both, into the gel. Cells grown with laminin showed the best proliferation. Cells grown with RGDSP proliferated a few times and then started to spread out. Cells grown with the combination of RGDSP and laminin showed better proliferation than with RGDSP alone and larger spread-outs than with laminin alone. After stimulations with first FGF2 and EGF, and then only FGF2, some cells showed neuronal morphology after 2 weeks. The neuronal population was assessed by the presence of neuronal marker b-tubulin-III. Glial cells were also present. Further characterizations are undergoing. Conclusions: RSC can grow and migrate in 3D hydrogel with the addition of FGF2, EGF, RGDSP and laminin. Further developments are necessary to form a homogenous tissue containing retinal cells.

EZH2 is essential for glioblastoma cancer stem cell maintenance.

Overexpression of the polycomb group protein enhancer of zeste homologue 2 (EZH2) occurs in diverse malignancies, including prostate cancer, breast cancer, and glioblastoma multiforme (GBM). Based on its ability to modulate transcription of key genes implicated in cell cycle control, DNA repair, and cell differentiation, EZH2 is believed to play a crucial role in tissue-specific stem cell maintenance and tumor development. Here, we show that targeted pharmacologic disruption of EZH2 by the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep), or its specific downregulation by short hairpin RNA (shRNA), strongly impairs GBM cancer stem cell (CSC) self-renewal in vitro and tumor-initiating capacity in vivo. Using genome-wide expression analysis of DZNep-treated GBM CSCs, we found the expression of c-myc, recently reported to be essential for GBM CSCs, to be strongly repressed upon EZH2 depletion. Specific shRNA-mediated downregulation of EZH2 in combination with chromatin immunoprecipitation experiments revealed that c-myc is a direct target of EZH2 in GBM CSCs. Taken together, our observations provide evidence that direct transcriptional regulation of c-myc by EZH2 may constitute a novel mechanism underlying GBM CSC maintenance and suggest that EZH2 may be a valuable new therapeutic target for GBM management.

'Hearts and bones': the ups and downs of 'plasticity' in stem cell biology.

More than a decade ago, 'plasticity' suddenly became a 'fashionable' topic with overemphasized implications for regenerative medicine. The concept of 'plasticity' is supported by old transplantation work, at least for embryonic cells, and metaplasia is a classic example of plasticity observed in patients. Nevertheless, the publication of a series of papers showing rare conversion of a given cell type into another unrelated cell raised the possibility of using any unaffected tissue to create at will new cells to replace a different failing tissue or organ. This resulted in disingenuous interpretations and a reason not to fund anymore research on embryonic stem cells (ESc). Moreover, many papers on plasticity were difficult to reproduce and thus questioned; raising issues about plasticity as a technical artefact or a consequence of rare spontaneous cells fusion. More recently, reprogramming adult differentiated cells to a pluripotent state (iPS) became possible, and later, one type of differentiated cell could be directly reprogrammed into another (e.g. fibroblasts into neurons) without reverting to pluripotency. Although the latter results from different and more robust experimental protocols, these phenomena also exemplify 'plasticity'. In this review, we want to place 'plasticity' in a historical perspective still taking into account ethical and political implications.

Dormant and self-renewing hematopoietic stem cells and their niches.

In the mouse, over the last 20 years, a set of cell-surface markers and activities have been identified, enabling the isolation of bone marrow (BM) populations highly enriched in hematopoietic stem cells (HSCs). These HSCs have the ability to generate multiple lineages and are capable of long-term self-renewal activity such that they are able to reconstitute and maintain a functional hematopoietic system after transplantation into lethally irradiated recipients. Using single-cell reconstitution assays, various marker combinations can be used to achieve a functional HSC purity of almost 50%. Here we have used the differential expression of six of these markers (Sca1, c-Kit, CD135, CD48, CD150, and CD34) on lineage-depleted BM to refine cell hierarchies within the HSC population. At the top of the hierarchy, we propose a dormant HSC population (Lin(-)Sca1(+)c-Kit(+) CD48(-)CD150(+)CD34(-)) that gives rise to an active self-renewing CD34(+) HSC population. HSC dormancy, as well as the balance between self-renewal and differentiation activity, is at least, in part, controlled by the stem cell niches individual HSCs are attached to. Here we review the current knowledge about HSC niches and propose that dormant HSCs are located in niches at the endosteum, whereas activated HSCs are in close contact to sinusoids of the BM microvasculature.

Hard-wired heterogeneity in blood stem cells revealed using a dynamic regulatory network model.

MOTIVATION: Combinatorial interactions of transcription factors with cis-regulatory elements control the dynamic progression through successive cellular states and thus underpin all metazoan development. The construction of network models of cis-regulatory elements, therefore, has the potential to generate fundamental insights into cellular fate and differentiation. Haematopoiesis has long served as a model system to study mammalian differentiation, yet modelling based on experimentally informed cis-regulatory interactions has so far been restricted to pairs of interacting factors. Here, we have generated a Boolean network model based on detailed cis-regulatory functional data connecting 11 haematopoietic stem/progenitor cell (HSPC) regulator genes. RESULTS: Despite its apparent simplicity, the model exhibits surprisingly complex behaviour that we charted using strongly connected components and shortest-path analysis in its Boolean state space. This analysis of our model predicts that HSPCs display heterogeneous expression patterns and possess many intermediate states that can act as 'stepping stones' for the HSPC to achieve a final differentiated state. Importantly, an external perturbation or 'trigger' is required to exit the stem cell state, with distinct triggers characterizing maturation into the various different lineages. By focusing on intermediate states occurring during erythrocyte differentiation, from our model we predicted a novel negative regulation of Fli1 by Gata1, which we confirmed experimentally thus validating our model. In conclusion, we demonstrate that an advanced mammalian regulatory network model based on experimentally validated cis-regulatory interactions has allowed us to make novel, experimentally testable hypotheses about transcriptional mechanisms that control differentiation of mammalian stem cells. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling.

Continuous turnover of epithelia is ensured by the extensive self-renewal capacity of tissue-specific stem cells. Similarly, epithelial tumour maintenance relies on cancer stem cells (CSCs), which co-opt stem cell properties. For most tumours, the cellular origin of these CSCs and regulatory pathways essential for sustaining stemness have not been identified. In murine skin, follicular morphogenesis is driven by bulge stem cells that specifically express CD34. Here we identify a population of cells in early epidermal tumours characterized by phenotypic and functional similarities to normal bulge skin stem cells. This population contains CSCs, which are the only cells with tumour initiation properties. Transplants derived from these CSCs preserve the hierarchical organization of the primary tumour. We describe beta-catenin signalling as being essential in sustaining the CSC phenotype. Ablation of the beta-catenin gene results in the loss of CSCs and complete tumour regression. In addition, we provide evidence for the involvement of increased beta-catenin signalling in malignant human squamous cell carcinomas. Because Wnt/beta-catenin signalling is not essential for normal epidermal homeostasis, such a mechanistic difference may thus be targeted to eliminate CSCs and consequently eradicate squamous cell carcinomas.

Bcl9/Bcl9l are critical for Wnt-mediated regulation of stem cell traits in colon epithelium and adenocarcinomas.

Canonical Wnt signaling plays a critical role in stem cell maintenance in epithelial homeostasis and carcinogenesis. Here, we show that in the mouse this role is critically mediated by Bcl9/Bcl9l, the mammalian homologues of Legless, which in Drosophila is required for Armadillo/beta-catenin signaling. Conditional ablation of Bcl9/Bcl9l in the intestinal epithelium, where the essential role of Wnt signaling in epithelial homeostasis and stem cell maintenance is well documented, resulted in decreased expression of intestinal stem cell markers and impaired regeneration of ulcerated colon epithelium. Adenocarcinomas with aberrant Wnt signaling arose with similar incidence in wild-type and mutant mice. However, transcriptional profiles were vastly different: Whereas wild-type tumors displayed characteristics of epithelial-mesenchymal transition (EMT) and stem cell-like properties, these properties were largely abrogated in mutant tumors. These findings reveal an essential role for Bcl9/Bcl9l in regulating a subset of Wnt target genes involved in controlling EMT and stem cell-related features and suggest that targeting the Bcl9/Bcl9l arm of Wnt signaling in Wnt-activated cancers might attenuate these traits, which are associated with tumor invasion, metastasis, and resistance to therapy.

Cardiac dysfunction and impaired compensatory response to pressure overload in mice deficient in stem cell antigen-1.

Stem cell antigen-1 (Sca-1) has been used to identify cardiac stem cells in the mouse heart. To investigate the function of Sca-1 in aging and during the cardiac adaptation to stress, we used Sca-1-deficient mice. These mice developed dilated cardiomyopathy [end-diastolic left ventricular diameter at 18 wk of age: wild-type (WT) mice, 4.2 mm ± 0.3; Sca-1-knockout (Sca-1-KO) mice, 4.6 mm ± 0.1; ejection fraction: WT mice, 51.1 ± 2.7%; Sca-1-KO mice, 42.9 ± 2.7%]. Furthermore, the hearts of mice lacking Sca-1 demonstrated exacerbated susceptibility to pressure overload [ejection fraction after transaortic constriction (TAC): WT mice, 43.5 ± 3.2%; Sca-1-KO mice, 30.8% ± 4.0] and increased apoptosis, as shown by the 2.5-fold increase in TUNEL(+) cells in Sca-1-deficient hearts under stress. Sca-1 deficiency affected primarily the nonmyocyte cell fraction. Indeed, the number of Nkx2.5(+) nonmyocyte cells, which represent a population of cardiac precursor cells (CPCs), was 2-fold smaller in Sca-1 deficient neonatal hearts. In vitro, the ability of CPCs to differentiate into cardiomyocytes was not affected by Sca-1 deletion. In contrast, these cells demonstrated unrestricted differentiation into cardiomyocytes. Interestingly, proliferation of cardiac nonmyocyte cells in response to stress, as judged by BrdU incorporation, was higher in mice lacking Sca-1 (percentages of BrdU(+) cells in the heart after TAC: WT mice, 4.4 ± 2.1%; Sca-1-KO mice, 19.3 ± 4.2%). These data demonstrate the crucial role of Sca-1 in the maintenance of cardiac integrity and suggest that Sca-1 restrains spontaneous differentiation in the precursor population. The absence of Sca-1 results in uncontrolled precursor recruitment, exhaustion of the precursor pool, and cardiac dysfunction.

Applications of flow cytometry to hematopoietic stem cell transplantation

Applications of flow cytometry to clinical and experimental hematopoietic stem cell transplantation (HSCT) are discussed in this review covering the following topics: diagnosis and classification of lymphohematologic disorders, quantitation of hematopoietic progenitors in the graft, lymphohematopoietic reconstitution following HSCT and animal models of human HSCT. At the end, the utilization of flow cytometry in clinical HSCT by Brazilian transplant centers is briefly reviewed.

Breast stem cells and hormonal mechanisms in carcinogenesis

A woman's risk of breast cancer is strongly affected by her reproductive history. The hormonal milieu is also a key determinant of the course of the disease. Combining mouse genetics with tissue recombination techniques, we have established that the female reproductive hormones, estrogens, progesterone, and prolactin, act sequentially on the mammary epithelium to trigger distinct developmental steps. The hormones impinge directly on a subset of luminal mammary epithelial cells that express the respective hormone receptors and act as sensor cells translating and amplifying systemic signals into local stimuli. Local signaling is stage and age specific. During puberty, estrogens promote proliferation using the EGF family member, amphiregulin, as essential paracrine mediator. In adulthood, progesterone, rather than estrogen, is the major inducer of stem cell activation and cell proliferation of the mammary epithelium. Hormonal signaling modulates crucial developmental pathways that impinge on mammary stem cell populations, while Notch signaling, by inhibiting p63, is central to mammary cell fate determination. Cell proliferation occurs in two waves. The first results from direct stimulation of the small fraction of hormone receptor positive cells. It is followed by a second wave of progesterone-induced proliferation involving mostly hormone receptor negative cells, in which RANKL is a key mediator. A model in which repeated activation of paracrine signaling by progesterone with resulting stem cell activation promotes breast carcinogenesis is proposed.

Prenatally engineered autologous amniotic fluid stem cell-based heart valves in the fetal circulation.

Prenatal heart valve interventions aiming at the early and systematic correction of congenital cardiac malformations represent a promising treatment option in maternal-fetal care. However, definite fetal valve replacements require growing implants adaptive to fetal and postnatal development. The presented study investigates the fetal implantation of prenatally engineered living autologous cell-based heart valves. Autologous amniotic fluid cells (AFCs) were isolated from pregnant sheep between 122 and 128 days of gestation via transuterine sonographic sampling. Stented trileaflet heart valves were fabricated from biodegradable PGA-P4HB composite matrices (n = 9) and seeded with AFCs in vitro. Within the same intervention, tissue engineered heart valves (TEHVs) and unseeded controls were implanted orthotopically into the pulmonary position using an in-utero closed-heart hybrid approach. The transapical valve deployments were successful in all animals with acute survival of 77.8% of fetuses. TEHV in-vivo functionality was assessed using echocardiography as well as angiography. Fetuses were harvested up to 1 week after implantation representing a birth-relevant gestational age. TEHVs showed in vivo functionality with intact valvular integrity and absence of thrombus formation. The presented approach may serve as an experimental basis for future human prenatal cardiac interventions using fully biodegradable autologous cell-based living materials.

Cutting edge: influence of the TCR Vbeta domain on the selection of semi-invariant NKT cells by endogenous ligands.

Invariant Valpha14 (Valpha14i) NKT cells are a murine CD1d-dependent regulatory T cell subset characterized by a Valpha14-Jalpha18 rearrangement and expression of mostly Vbeta8.2 and Vbeta7. Whereas the TCR Vbeta domain influences the binding avidity of the Valpha14i TCR for CD1d-alpha-galactosylceramide complexes, with Vbeta8.2 conferring higher avidity binding than Vbeta7, a possible impact of the TCR Vbeta domain on Valpha14i NKT cell selection by endogenous ligands has not been studied. In this study, we show that thymic selection of Vbeta7(+), but not Vbeta8.2(+), Valpha14i NKT cells is favored in situations where endogenous ligand concentration or TCRalpha-chain avidity are suboptimal. Furthermore, thymic Vbeta7(+) Valpha14i NKT cells were preferentially selected in vitro in response to CD1d-dependent presentation of endogenous ligands or exogenously added self ligand isoglobotrihexosylceramide. Collectively, our data demonstrate that the TCR Vbeta domain influences the selection of Valpha14i NKT cells by endogenous ligands, presumably because Vbeta7 confers higher avidity binding.