Phenotypic and functional analysis of human fetal liver hematopoietic stem cells in culture.

Steady-state hematopoiesis and hematopoietic transplantation rely on the unique potential of stem cells to undergo both self-renewal and multilineage differentiation. Fetal liver (FL) represents a promising alternative source of hematopoietic stem cells (HSCs), but limited by the total cell number obtained in a typical harvest. We reported that human FL nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) could be expanded under simple stroma-free culture conditions. Here, we sought to further characterize FL HSC/SRCs phenotypically and functionally before and following culture. Unexpanded or cultured FL cell suspensions were separated into various subpopulations. These were tested for long-term culture potential and for in vivo repopulating function following transplantation into NOD/SCID mice. We found that upon culture of human FL cells, a tight association between classical stem cell phenotypes, such as CD34(+) /CD38(-) and/or side population, and NOD/SCID repopulating function was lost, as observed with other sources. Although SRC activity before and following culture consistently correlated with the presence of a CD34(+) cell population, we provide evidence that, contrary to umbilical cord blood and adult sources, stem cells present in both CD34(+) and CD34(-) FL populations can sustain long-term hematopoietic cultures. Furthermore, upon additional culture, CD34-depleted cell suspensions, devoid of SRCs, regenerated a population of CD34(+) cells possessing SRC function. Our studies suggest that compared to neonatal and adult sources, the phenotypical characteristics of putative human FL HSCs may be less strictly defined, and reinforce the accumulated evidence that human FL represents a unique, valuable alternative and highly proliferative source of HSCs for clinical applications.

Mobilization of hemopoietic stem cells with high-dose methotrexate plus granulocyte-colony-stimulating factor in patients with primary central nervous system lymphoma.

BACKGROUND: High-dose therapy with autologous stem cell support after standard dose induction is a promising approach for therapy of primary central nervous system lymphoma (PCNSL). High-dose methotrexate (HD-MTX) is a standard drug for induction of PCNSL; however, data about the capacity of HD-MTX plus granulocyte-colony-stimulating factor (G-CSF) to mobilize hemopoietic progenitors are lacking. STUDY DESIGN AND METHODS: This investigation describes the data from stem cell mobilization and apheresis procedures after one or two cycles of HD-MTX for induction of PCNSL within the East German Study Group for Haematology and Oncology 053 trial. Eligible patients proceeded to high-dose busulfan/thiotepa after induction therapy and mobilization. RESULTS: Data were available from nine patients with a median age of 58 years. The maximal CD34+ cell count per microL of blood after the first course of HD-MTX was 13.89 (median). Determination was repeated in six patients after the second course with a significantly higher median CD34+ cell count of 33.69 per microL. Five patients required two apheresis procedures and in four patients a single procedure was sufficient. The total yield of CD34+ cells per kg of body weight harvested by one or two leukapheresis procedures was 6.60 x 10(6) (median; range, 2.68 x 10(6)-15.80 x 10(6)). The yield of CD34+ cells exceeded the commonly accepted lower threshold of 3 x 10(6) cells per kg of body weight in eight of nine cases. Even in the ninth, hemopoietic recovery after stem cell reinfusion was rapid and safe. CONCLUSION: HD-MTX plus G-CSF is a powerful combination for stem cell mobilization in patients with PCNSL and permits safe conduction of time-condensed and dose-intense protocols with high-dose therapy followed by stem cell reinfusion after HD-MTX induction.

Limited telomere shortening in hematopoietic stem cells after transplantation.

The number of cell divisions in hematopoietic stem cells (HSCs) following transplantation of bone marrow or mobilized peripheral blood into myelo-ablated recipients is unknown. This number is expected to depend primarily on the number of transplanted stem cells, assuming that stem cells do not differ in engraftment potential and other functional properties. In a previous study, we found that the telomere length in circulating granulocytes in normal individuals shows a biphasic decline with age, most likely reflecting age-related changes in the turnover of HSCs. In order to study HSCs' proliferation kinetics following stem cells transplantation, we analyzed the telomere length in donor-derived nucleated blood cells in four HLA-matched bone marrow transplant recipients relative to comparable cells from the sibling donors. In each case, the telomeres in granulocytes were shorter in the recipient than in the donor. This difference was established in the first year post transplantation and did not change after that. The telomere length in naïve and memory T cells showed marked differences after transplantation, complicating the interpretation of telomere length data using unseparated nucleated blood cells. Interestingly, the telomere length in naïve T cells that were first observed six months post transplantation was very similar in donor and recipient pairs. Our observations are compatible with a limited number of additional cell divisions in stem cell populations after bone marrow transplantations and support the idea that different populations of stem cells contribute to short-term myeloid and long-term lympho myeloid hematopoiesis.

Inhibition of Notch Pathway Enhances Photoreceptor Commitment From Cultured Retinal Stem Cells

Purpose: Consequently to the principle that photoreceptors have to be at a very precise development stage to be successfully transplanted (MacLaren 2006), we are trying to mimic this development stage in vitro using retinal stem cells. The latter one isolated from the newborn mouse retina, derived from the radial glia population, which were previously isolated and characterized in our laboratory. We developed a protocol to commit these cells to the photoreceptor fate, but even if the percentage of cells expressing photoreceptor markers is high (30%), the differentiation process is incomplete so far (Merhi-Soussi 2006). Methods: In order to ameliorate photoreceptor differentiation, we hypothesized that the Notch pathway may interfere with this process by either promoting glia commitment, or maintaining an undifferentiated state. We are thus using a gamma-secretase inhibitor (DAPT), which inhibits Notch receptor cleavage and thus Notch activation. DAPT was used either during the whole differentiation stimulation, or only during a restricted period in two various retinal stem cell lines (RSC AA and RSC MP1). Results: RT-PCR performed during cell proliferation, showed the same positive expression in both cell lines for the following genes: Math3, Six3, Hes1, NeuroD, Pax6 and Notch1. Additionally, Mash1, Hes5, Prox1, Crx and Otx2 were detected in both cell lines but with a stronger expression in RSC MP1. Opposite results were obtained for Chx10. Nrl, Peripherin/RDS, GFAP and Math5 were detected neither in RSC AA, nor in RSC MP1. The constant presence of DAPT i) leads to a 233% (RSC AA) or 900% (RSC MP1) increase in peripherin/RDS-positive (photoreceptor marker) cells, compared to controls (no DAPT, n=3, P<0.02) along with a 68% (RSC AA) or 80% (RSC MP1) decrease in GFAP- positive cells (n=3, P<0.04), ii) modifies the ratio between uni-/bi- (23%) and multi- (77%) polar peripherin/RDS-positive cells to 45% and 55%, respectively, for both cell lines and iii) reduces by 50% the total cell number during the whole differentiation process for both cell lines. Conclusions: We are now exploring whether this reduction in total cell number is due to inhibition of cell proliferation or to cell death and whether photoreceptor differentiation is promoted instead of glial induction. We also want to confirm the results obtained with DAPT with RSCs isolated from Notch1-loxP mice. Such protocol may help to better mimic photoreceptor development, but this needs to be confirmed by genomic and proteomic profile analyses.

Caspofungin first-line therapy for invasive aspergillosis in allogeneic hematopoietic stem cell transplant patients: an European Organisation for Research and Treatment of Cancer study.

Caspofungin at standard dose was evaluated as first-line monotherapy of mycologically documented probable/proven invasive aspergillosis (IA) (unmodified European Organisation for Research and Treatment of Cancer/Mycosis Study Group criteria) in allogeneic hematopoietic SCT patients. The primary efficacy end point was complete or partial response at end of caspofungin treatment. Response at week 12, survival and safety were additional end points. Enrollment was stopped prematurely because of low accrual, with 42 enrolled and 24 eligible, giving the study a power of 85%. Transplant was from unrelated donors in 16 patients; acute or chronic GVHD was present in 15. In all, 12 patients were neutropenic (<500/microl) at baseline, 10 received steroids and 16 calcineurin inhibitors or sirolimus. Median duration of caspofungin treatment was 24 days. At the end of caspofungin therapy, 10 (42%) patients had complete or partial response (95% confidence interval: 22-63%); 1 (4%) and 12 (50%) had stable and progressing disease, respectively; one was not evaluable. At week 12, eight patients (33%) had complete or partial response. Survival rates at week 6 and 12 were 79 and 50%, respectively. No patient had a drug-related serious adverse event or discontinued because of toxicity. Caspofungin first-line therapy was effective and well tolerated in allogeneic hematopoietic SCT patients with mycologically documented IA.

Consolidation with Radioimmunotherapy May Prolong Survival in First Remission of Mantle Cell Lymphoma Patients Uneligible for Stem Cell Transplantation, An Analysis of the International RIT-Network

Background: Mantle cell lymphoma (MCL) is a rare subtype (3-9%) of Non Hodgkin Lymphoma (NHL) with a relatively poor prognosis (5-year survival < 40%). Although consolidation of first remission with autologous stem cell transplantation (ASCT) is regarded as "golden standard", less than half of the patients may be subjected to this intensive treatment due to advanced age and co-morbidities. Standard-dose non-myeloablative radioimmunotherapy (RIT) seems to be a very efficient approach for treatment of certain NHL. However, there are almost no data available on the efficacy and safety of RIT in MCL. Methods and Patients: In the RIT-Network, a web-based international registry collecting real observational data from RIT-treated patients, 115 MCL patients treated with ibritumomab tiuxetan were recorded. Most of the patients were elderly males with advanced stage of the disease: median age - 63 (range 31-78); males - 70.4%, stage III/IV - 92%. RIT (i.e. application of ibritumomab tiuxetan) was a part of the first line therapy in 48 pts. (43%). Further 38 pts. (33%) received ibritumomab tiuxetan after two previous chemotherapy regimens, and 33 pts. (24%) after completing 3-8 lines. In 75 cases RIT was applied as a consolidation of chemotherapy induced response; the rest of the patients received ibritumomab tiuxetan because of relapse/refractory disease. At the moment follow up data are available for 74 MCL patients. Results: After RIT the patients achieved high response rate: CR 60.8%, PR 25.7%, and SD 2.7%. Only 10.8% of the patients progressed. For survival analysis many data had to be censored since the documentation had not been completed yet. The projected 3-year overall survival (OAS, fig.1 - image 001.gif) after radioimmunotherapy was 72% for pts. subjected to RIT consolidation versus 29% for those treated in relapse/refractory disease (p=0.03). RIT was feasible for almost all patients; only 3 procedure-related deaths were reported in the whole group. The main adverse event was hematological toxicity (grade III/IV cytopenias) showing a median time of recovery of Hb, WBC and Plt of 45, 40 and 38 days respectively. Conclusion: Standard-dose non-myeloablative RIT is a feasible and safe treatment modality, even for elderly MCL pts. Consolidation radioimmunotherapy with ibritumomab tiuxetan may prolong survival of patients who achieved clinical response after chemotherapy. Therefore, this consolidation approach should be considered as a treatment strategy for those, who are not eligible for ASCT. RIT also has a potential role as a palliation therapy in relapsing/resistant cases.

Awakening dormant haematopoietic stem cells.

Haematopoietic stem cells (HSCs) in mouse bone marrow are located in specialized niches as single cells. During homeostasis, signals from this environment keep some HSCs dormant, which preserves long-term self-renewal potential, while other HSCs actively self renew to maintain haematopoiesis. In response to haematopoietic stress, dormant HSCs become activated and rapidly replenish the haematopoietic system. Interestingly, three factors - granulocyte colony-stimulating factor, interferon-alpha and arsenic trioxide - have been shown to efficiently activate dormant stem cells and thereby could break their resistance to anti-proliferative chemotherapeutics. Thus, we propose that two-step strategies could target resistant leukaemic stem cells by priming tumours with activators of dormancy followed by chemotherapy or targeted therapies.

3D culture of postnatal retinal stem cells using self assembling polymers

PURPOSE We have previously shown that retinal stem cells (RSCs) can be isolated from the radial glia population of the newborn mouse retina (Angénieux et al., 2006). These RSCs have a great capacity to renew and to generate a large number of neurons including cells differentiated towards the photoreceptor lineage (Mehri-Soussi et al., 2006). However, recent published results from our lab revealed that such cells have a poor integration and survival rate after grafting. The uncontrolled environment of a retina seems to prevent good integration and survival after grafting in vivo. To bypass this problem, we are evaluating the possibility of generating in vitro a hemi-retinal tissue before transplantation. METHODS RSC were expanded and cells passaged <10 were seeded in a solution containing poly-ethylene-glycol (PEG) polymer based hydrogels crosslinked with peptides that are chosen to be substrates for matrix metalloproteinases. Various doses of cross linkers peptides allowing connections between PEG polymers were tested. Different growth factors were studied to stimulate cell proliferation and differentiation. RESULTS Cells survived only in the presence of EGF and FGF-2 and generated colonies with a sphere shape. No cells migrated within the gel. To improve the migration and the repartition of the cells in the gels, the integrin ligand RGDSP was added into the gel. In the presence of FGF-2 and EGF, newly formed cell clusters appeared by cell proliferation within several days, but again no outspreading of cells was observed. No difference was even seen when the stiffness of the hydrogels or the concentration of the integrin ligand RGDSP were changed. However, our preliminary results show that RSCs still form spheres when laminin is entrapped in the gel, but they started to spread out having a neuronal morphology after around 2 weeks. The neuronal population was assessed by the presence of the neuronal marker b-tubulin-III. This differentiation was achieved after successive steps of stimulations including FGF-2 and EGF, and then only FGF-2. Glial cells were also present. Further characterizations are under process. CONCLUSIONS RSC can be grown in 3D. Preliminary results show that neuronal cell phenotype acquisition can be instructed by exogenous stimulations and factors linked to the gel. Further developments are necessary to form a homogenous tissue containing retinal cells.

Smooth height/age curves from stem analysis with linear programming

[Abstract]

Effect of thinning on stem form and wood characteristics of teak (Tectona grandis) in a humid tropical site in Costa Rica

Abstract

Differential miRNA and IncRNA expression in embryonic stem cells lacking the Notch1 receptor

Embryonic stem (ES) cells-derived cardiomyocytes represent an attractive source of cells in cell replacement therapies for heart disease. However, controlled cardiogenic differentiation of ES cells requires a complete understanding of the complex molecular mechanisms regulating the differentiation process. We have previously shown that differentiation of ES cells into cardiomyocytes is favored by inactivation of the Notch 1 receptor pathway. In the present study, we therefore compared two ES cell lines, one with normal Notchl expression and one carrying deleted Notchl receptor alleles (Notchl-deleted ES cells) in order to identify genes responsible for the increased propensity of Notchl-deleted ES cells to produce cardiomyocytes. Using RNA-sequencing, we found approximately 300 coding and noncoding transcripts, which are differently expressed in undifferentiated Notchl-deleted ES cells. Since accumulating evidences indicate that long noncoding RNAs (IncRNAs) play important roles in ES cell pluripotency and differentiation, we focused our analysis on modulated IncRNAs. In particular, two IncRNAs, named here lnc 1230 and lnc 1335, are highly induced in the absence of Notchl receptor expression. These represent therefore prime candidates that could favor cardiogenic commitment in undifferentiated ES cells. Indeed, we demonstrate that forced expression of these two IncRNAs in wild-type ES cells result in a significant increase of the number of cardiac progenitor cells and cardiomyocytes in the differentiated progeny of these ES cells. Furthermore, we also identify several microRNAs that are differentially modulated in absence of Notchl expression. Among these are miR-142-5p and miR- 381-3p. Interestingly, both lncl230 and lncl335 are targets of these two microRNAs. Altogether, these data suggest that Notchl-dependent noncoding gene networks, implicating microRNAs and IncRNAs, control embryonic stem cell commitment into the mesodermal and cardiac lineages already at the undifferentiated state. - Les cardiomyocytes issus cellules souches embryonnaires sont une source très prometteuse pour les thérapies cellulaire de remplacement dans le cadre des maladies cardiaques. Cependant, l'utilisation de telles cellules requiert une compréhension poussée des mécanismes moléculaire régulant la différenciation. Nous avons par le passé démontré que la différenciation des cellules souches embryonnaires en cardiomyocytes est favorisée par l'inactivation de la voie d'activation intracellulaire dépendante du récepteur Notch 1. Nous avons donc comparé deux lignées de cellules souches embryonnaires, une présentant une voie d'activation Notchl normale et une chez laquelle les allèles codant pour le récepteur Notchl avaient été invalidés, de façon à identifier les gènes impliqués dans la capacité augmentée des cellules déficientes à produire des cardiomyocytes. En utilisant du séquençage d'ARN à haut débit, nous avons trouvé environ 300 gènes différemment exprimés dans les cellules déficientes pour Notchl. Par ailleurs, des évidences de plus en plus nombreuses suggèrent qu'une nouvelle classe de molécules appelée « long noncoding RNAs » joue un rôle prépondérant dans la maintenance de l'état non différencié et de la capacité de différenciation des cellules souches embryonnaires. Nous avons trouvé que plusieurs « long noncoding RNAs » étaient modulés en l'absence de Notchl, et en particulier deux molécules que nous avons appelées lncl230 et lncl335. Ces derniers représentent des candidats potentiels devant permettre de favoriser la production de cardiomyocytes. Nous avons en effet démontré que la surexpression de ces deux candidats dans des cellules souches embryonnaires résultait en une surproduction de cardiomyocytes. De plus, nous avons également identifié plusieurs microRNAs dont l'expression était modulée dans les cellules souches embryonnaires déficientes dans la voie Notchl. De façon intéressante, parmi ces microRNAs, le miR-142-5p et le miR-381-3p sont capables de cibler lncl230 and lncl335. Dans l'ensemble, ces résultats indiquent donc que des réseaux d'interaction dépendant de la voie d'activation Notch 1 et impliquant des ARNs non codant existent dans les cellules souches embryonnaires pour réguler leur différenciation en différent types cellulaires spécifiques.

Telomere length dynamics in normal individuals and in patients with hematopoietic stem cell-associated disorders.

The telomere length in nucleated peripheral blood (PB) cells indirectly reflects the mitotic history of their precursors: the hematopoietic stem cells (HSCs). The average length of telomeres in PB leukocytes can be measured using fluorescence in situ hybridization and flow cytometry (flow FISH). We previously used flow FISH to characterize the age-related turnover of HSCs in healthy individuals. In this review, we describe results of recent flow FISH studies in patients with selected hematopoietic stem cell-associated disorders: chronic myelogenous leukemia (CML) and several bone marrow failure syndromes. CML is characterized by a marked expansion of myeloid Philadelphia chromosome positive (Ph+) cells. Nevertheless, nonmalignant (Ph-) HSCs typically coexist in the bone marrow of CML patients. We analyzed the telomere length in > 150 peripheral blood leukocytes (PBLs) and bone marrow samples of patients with CML as well as samples of Ph- T-lymphocytes. Compared to normal controls, the overall telomere fluorescence in PBLs of patients with CML was significantly reduced. However, no telomere shortening was observed in Ph- T-lymphocytes. Patients in late chronic phase (CP) had significantly shorter telomeres than those assessed earlier in CP. Our data suggest that progressive telomere shortening is correlated with disease progression in CML. Within the group of patients with bone marrow failure syndromes, we only found significantly shortened telomeres (compared to age-adjusted controls) in granulocytes from patients with aplastic anemia (AA). Strikingly, the telomere length in granulocytes from AA patients who had recovered after immunosuppressive therapy (recAA) did not differ significantly from controls, whereas untreated patients and nonresponders with persistent severe pancytopenia (sAANR) showed marked and significant telomere shortening compared to healthy donors and patients with recAA. Furthermore, an inverse correlation between age-adjusted telomere length and peripheral blood counts was found in support of a model in which the degree of cytopenia and the amount of telomere shortening are correlated. These results support the concept of extensive proliferation of HSCs in subgroups of AA patients and suggest a potential use of telomere-length measurements as a prognostic tool in this group of disorders as well.

c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation.

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Here, we provide genetic evidence for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, our data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche.

Derivation of traceable and transplantable photoreceptors from mouse embryonic stem cells.

Retinal degenerative diseases resulting in the loss of photoreceptors are one of the major causes of blindness. Photoreceptor replacement therapy is a promising treatment because the transplantation of retina-derived photoreceptors can be applied now to different murine retinopathies to restore visual function. To have an unlimited source of photoreceptors, we derived a transgenic embryonic stem cell (ESC) line in which the Crx-GFP transgene is expressed in photoreceptors and assessed the capacity of a 3D culture protocol to produce integration-competent photoreceptors. This culture system allows the production of a large number of photoreceptors recapitulating the in vivo development. After transplantation, integrated cells showed the typical morphology of mature rods bearing external segments and ribbon synapses. We conclude that a 3D protocol coupled with ESCs provides a safe and renewable source of photoreceptors displaying a development and transplantation competence comparable to photoreceptors from age-matched retinas.