scholarly journals Imetelstat Inhibits Telomerase and Prevents Propagation of ADAR1-Activated Myeloproliferative Neoplasm and Leukemia Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-18
Author(s):  
Wenxue Ma ◽  
Larissa Balaian ◽  
Phoebe Mondala ◽  
Yudou He ◽  
Cayla Mason ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Telomere Length ◽  
Mouse Models ◽  
Telomerase Activity ◽  
Rna Seq ◽  
Publicly Traded ◽  
Cd34 Cells

BACKGROUND Clonal stem cell derived myeloproliferative neoplasms (MPNs) have a propensity to evolve to acute myeloid leukemia (AML). Deregulation of the innate immune deaminase associated with RNA1 (ADAR1) has been linked to malignant progression and therapeutic resistance. Increased expression of the stem cell gene, human telomerase reverse transcriptase (hTERT), has also been linked with malignant transformation. However, the combinatorial role of ADAR1 and hTERT in the evolution of MPN stem cells to therapy resistant acute myeloid leukemia stem cells (LSCs) and the capacity of a telomerase inhibitor, imetelstat, to prevent survival and self-renewal of pre-LSC and LSC had not been established. Recent clinical trials show early signs of efficacy of imetelstat in treatment of myelofibrosis (MF). However, its role in selectively inhibiting pre-LSC transformation to self-renewing LSC has not been elucidated. Here we show that targeting telomerase activity prevents pre-LSC and LSC maintenance both in vitro and in vivo, suggesting telomerase inhibition as an effective strategy for preventing MPN progression. METHODS To quantify hTERT level and ADAR1 activity in the setting of normal HSPC and MPN stem cell evolution, whole genome sequencing (WGS) analysis was performed on 76 normal and MPN blood CD34+ cells and matching saliva samples. Results were compared with RNA-seq of 100 FACS purified young, aged, MPN and AML CD34+CD38- stem cells and CD34+CD38+ progenitor cells. Confocal fluorescence microscopic evaluation of stem cell ADAR1 and hTERT localization, telomere length by Flow-FISH and telomerase activity by TRAP assays, lentiviral ADAR1 overexpression and shRNA knockdown were performed. In vitro stromal co-cultures, and humanized immunocompromised mouse models were established to determine the impact of imetelstat (a oligonucleotide inhibitor of telomerase) on normal, MPN stem cell and LSC maintenance. RESULTS Combined hTERT overexpression, ADAR1 activation and a significant reduction in telomere length correlated with accelerated stem cell aging during MPN progression to AML. Increased ADAR1 mediated adenosine to inosine (A-to-I) transcript editing coincided with accelerated telomere shortening in high risk MPN stem cells. Moreover, lentiviral ADAR1 overexpression enhanced pre-LSC engraftment. Treatment with imetelstat reduced MPN stem cell and LSC propagation in stromal co-cultures as well as in humanized mouse models commensurate with reduced hTERT expression levels and telomerase activity and decreased ADAR1 editing activity. Specifically, stromal co-culture assays revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 µM or 5 µM significantly inhibited survival and replating of blast crisis (BC) CML progenitors compared with aged bone marrow progenitors (p < 0.001, ANOVA). As a single agent, imetelstat (5 µM) inhibited survival and replating of pre-LSC derived from myelofibrosis compared with normal bone marrow progenitor samples (p < 0.001, ANOVA). In pre-LSC MPN NSG-SGM mouse models established from 4 different MF samples, a significant reduction in proliferation of human CD45+ cells (p < 0.01, t test) was observed in bone marrow and spleen, when compared with vehicle control. Treatment of humanized LSC mouse models, established with 5 different BC CML, with 30 mg/kg of imetelstat, 3 times a week for 4 weeks resulted in a significant reduction in proliferation of malignant progenitors and human CD45+ cells (p < 0.001, ANOVA). As measured by a Flow-FISH assay, abnormal telomere length was reversed by imetelstat treatment compared with mismatch control (p < 0.05, ANOVA). In addition, FACS analysis revealed a significant reduction in activated beta-catenin expression after imetelstat treatment of LSC engrafted mice compared with vehicle control (p < 0.01, ANOVA). Finally, RNA-seq analysis performed on human CD34+ cells from imetelstat treated LSC mouse models revealed a significant reduction in LSC harboring malignant ADAR1-mediated A-to-I editing at doses that spared normal hematopoietic stem cells. CONCLUSIONS Combined WGS and RNA-Seq analyses, lentiviral ADAR1 overexpression, stromal co-culture assays and humanized pre-LSC and LSC mouse model studies reveal that pre-LSC evolution into LSC coincides with both ADAR1 and hTERT activation, which can be prevented with imetelstat. Disclosures Rizo: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Huang:Geron Corp: Current Employment, Current equity holder in publicly-traded company. Jamieson:Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals UM171 Regulates the Hematopoietic Differentiation of Human Acquired Aplastic Anemia-Derived Induced Pluripotent Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2500-2500
Author(s):  
Tellechea Maria Florencia ◽  
Flavia S. Donaires ◽  
Tiago C. Silva ◽  
Lilian F. Moreira ◽  
Yordanka Armenteros ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Patient Specific ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Induced Pluripotent

Aplastic anemia (AA) is characterized by a hypoplastic bone marrow associated with low peripheral blood counts. In acquired cases, the immune system promotes hematopoietic stem and progenitor cell (HSPC) depletion by the action of several pro-inflammatory Th1 cytokines. The current treatment options for severe cases consist of sibling-matched allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppressive therapy (IST) with anti-thymocyte globulin, cyclosporine, and eltrombopag. However, most patients are not eligible for HSCT and, although about 85% of patients respond to IST with eltrombopag, a proportion of patients eventually relapse, requiring further therapies. Failure to respond adequately to immunosuppression may be attributed to the scarcity of HSPCs at the time of diagnosis. Induced pluripotent stem cells (iPSCs) are potentially an alternative source of patient-specific hematopoietic cells. Patient-specific HSPCs derived from in vitro iPSC differentiation may serve as a tool to study the disease as well as a source of hematopoietic tissue for cell therapies. The pyrimidoindole molecule UM171 induces ex vivo expansion of HSCs of human cord and peripheral blood and bone marrow, but the pathways modulated by this molecule are not well understood. Here we evaluated the hematopoietic differentiation potential of iPSCs obtained from patients with acquired AA. We further determined the effects of UM171 on this differentiation process. First, we derived iPSCs from 3 patients with acquired AA after treatment (1 female; average age, 31 years; 2 partial responders, 1 complete responder) and 3 healthy subjects (3 females; average age, 61 years) and induced differentiation in vitro through the embryoid body system in cell feeder and serum-free medium supplemented with cytokines. The hematopoietic differentiation of healthy-iPSCs yielded 19% ± 8.1% (mean ± SEM) of CD34+cells after 16 days in culture, in contrast with 11% ± 4.9% of CD34+cells obtained from the differentiation of AA-iPSCs, which corresponds to a 1.7-fold reduction in CD34+cell yield. The total number of erythroid and myeloid CFUs was lower in the AA-iPSC group as compared to healthy-iPSCs (12±4.2 vs.24±7.2; respectively; p<0.03). These findings suggest that erythroid-derived AA-iPSC have an intrinsic defect in hematopoietic differentiation. Next, we tested whether UM171 modulated hematopoietic differentiation of AA-iPSCs. We found that UM171 significantly stimulated the differentiation of both healthy and AA-iPSCs. In the healthy-iPSC group, the percentage of CD34+cells was 1.9-fold higher when treated with UM171 compared to controls treated with DMSO (37% ± 7.8% vs.19% ± 8.1%; respectively; p<0.03) and in AA-iPSCs the increase was 3.9-fold (45% ± 11% vs. 11% ± 4.9%; p<0.07). The clonogenic capacity of progenitors to produce erythroid and myeloid colonies also was augmented in both groups in comparison to DMSO (28±11 vs. 23±7.2) for healthy-iPSCs and for AA-iPSCs (23±8.5 vs. 12±4.2, p<0.06). We then investigated the molecular pathways influenced by UM171. The transcriptional profile of differentiated CD34+cells showed that UM171 up-regulated genes involved in early hematopoiesis from mesoderm (BRACHYURY and MIXL1) and primitive streak specification (APELA and APLNR), to hemangioblasts and primitive hematopoietic progenitor commitment (TDGF1, SOX17, and KLF5). We also observed the up-regulation of pro-inflammatory NF-kB activators (MAP4K1, ZAP70, and CARD11) and the anti-inflammatory gene PROCR, a marker of cultured HSCs and an NF-kB inhibitor. This balanced network has been previously suggested to be modulated by UM171 (Chagraoui et. al. Cell Stem Cell 2019). Taken together, our results showed that acquired AA-iPSCs may have intrinsic defects that impair hematopoietic differentiation in vitro. This defect may be atavic to the cell or, alternatively, the consequence of epigenetic changes in erythroid precursors provoked by the immune attack. In addition, our findings demonstrate that UM171 significantly stimulate the hematopoietic differentiation of AA-iPSCs and identified a novel molecular mechanism for UM171 as an enhancer of early hematopoietic development programs. These observations may be valuable for improving the achievement of de novo hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

Telomerase activity, mTert gene expression and the telomere length in mouse mesenchymal stem cells in the late period after γ- and γ,n-irradiation and in the tumors developed from these cells

2020 ◽  
Vol 66 (3) ◽  
pp. 265-273
Author(s):  
O.V. Vysotskaya ◽  
A.I. Glukhov ◽  
Yu.P. Semochkina ◽  
S.A. Gordeev ◽  
E.Yu. Moskaleva
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Cell Lines ◽  
Telomerase Activity ◽  
Mouse Bone Marrow ◽  
Fibrosarcoma Cell

In proliferating normal and tumor cells, the telomere length (TL) is maintained by high telomerase activity (TA). In the absence of TA the TL maintenance involves a mechanism of alternative lengthening of telomeres (ALT). The aim of this study was to investigate the level of TA, the mTert expression and TL in cultured normal and transformed by γ- and γ,n-irradiation mesenchymal stem cells (MSCs) from mouse bone marrow, in sarcomas that developed after the transplantation of these cells into syngeneic mice, and in fibrosarcoma cell lines obtained from these tumors to find out the role of AT or ALT in maintaining TL in these cells. During prolonged cultivation of normal and transformed under the influence of γ- (1 Gy and 6 Gy) and γ,n-irradiation (0.05 Gy, 0.5 Gy, and 2 Gy) MSCs from mouse bone marrow, a decrease in TA was detected in irradiated cells. Even deeper decrease in TA was found in sarcomas developed after administration of transformed MSCs to syngeneic mice and in fibrosarcoma cell lines isolated from these tumors in which TA was either absent or was found to be at a very low level. TL in three of the four lines obtained was halved compared to the initial MSCs. With absent or low TA and reduced TL, the cells of all the obtained fibrosarcoma lines successfully proliferated without signs of a change in survival. The mechanism of telomere maintainance in fibrosarcoma cell lines in the absence of TA needs further investigation and it can be assumed that it is associated with the use of the ALT. The detected decrease or absence of TA in transformed under the action of irradiation MSCs with the preservation or even an increase in the telomerase gene expression may be associated with the formation of inactive splicing variants, and requires further study. The obtained lines of transformed MSCs and fibrosarcomas with TA and without the activity of this enzyme can be a useful model for studying the efficacy of TA and ALT inhibitors in vitro and in vivo.

Elevated telomerase activity and minimal telomere loss in cord blood long-term cultures with extensive stem cell replication

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4440-4448 ◽  
Author(s):  
Loretta Gammaitoni ◽  
Katja C. Weisel ◽  
Monica Gunetti ◽  
Kai-Da Wu ◽  
Stefania Bruno ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Telomere Length ◽  
Severe Combined Immunodeficiency ◽  
Telomerase Activity ◽  
Clinical Value ◽  
Telomere Shortening ◽  
Cd34 Cells

Abstract Telomerase activity, telomere length, stem/progenitor cell production, and function of CD34+ cells from cord blood (CB), bone marrow, and mobilized peripheral blood were evaluated in long-term cultures. CB cells were cultured either on OP-9 stromal cells transduced with an adenovector expressing thrombopoietin (TPO) or stimulated by a cytokine cocktail in the absence of stroma, with, in one method, CD34+ cells reisolated at monthly intervals for passage. Continuous expansion of stem cells as measured by in vitro cobblestone area and secondary colony-forming assays was noted for 18 to 20 weeks and by severe combined immunodeficiency (SCID)-repopulating cells (SRCs), capable of repopulating and serially passage in nonobese diabetic/SCID mice, for 16 weeks. Despite this extensive proliferation, telomere length initially increased and only at late stages of culture was evidence of telomere shortening noted. This telomere stabilization correlated with maintenance of high levels of telomerase activity in the CD34+ cell population for prolonged periods of culture. Cytokine-stimulated cultures of adult CD34+ cells showed CD34+ and SRC expansion (6-fold) for only 3 to 4 weeks with telomere shortening and low levels of telomerase. There is clearly a clinical value for a system that provides extensive stem cell expansion without concomitant telomere erosion. (Blood. 2004;103:4440-4448)

Long-Term Coculture of Adult Human Healthy Donor CD34+ Cells Shows Elevation of Telomeres in a Subgroup of Donors Independent of Age, Stem Cell Function and Telomerase Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1695-1695
Author(s):  
Katja C. Weisel ◽  
Kaida Wu ◽  
Lothar Kanz ◽  
Malcolm A.S. Moore
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Telomere Length ◽  
Healthy Donor ◽  
Progenitor Cell ◽  
Telomerase Activity ◽  
Suspension Cells ◽  
Telomere Shortening ◽  
Cd34 Cells

Abstract Long-term cytokine-supplemented or stromal cocultures of human CD34+ cells, particularly from cord blood (CB), show expansion of hematopoietic progenitors and stem cells. Ultimately, however cultures decline and terminally differentiate. Despite upregulation of telomerase activity in proliferating primitive hematopoietic cells, telomere shortening has generally been reported in long-term cultures of CB, bone marrow or G-CSF mobilized peripheral blood CD34+ cells. In earlier reports, we described a long-term culture of hematopoietic stem cells on a murine OP9 bone marrow stroma cell line transfected with an adenovector expressing thrombopoietin, which allowed an extensive proliferation and self-renewal of CB CD34+ cells for 4–5 months with sustained elevation of telomerase activity and without concomitant significant telomere shortening (Blood, 2004). Here, we evaluated adult healthy donor peripheral blood (PB) CD34+ cells in the same OP9/Tpo coculture system. To determine progenitor and stem cell production, standard CFC and 2ndry cobblestone area-forming cell assays (CAFC assayed at 5 weeks on MS5 stroma) were undertaken weekly with suspension cells. In addition telomere length was measured by telomere restriction fragment (TRF) assay, and telomerase activity by TRAP assay on input CD34+ cells, and weekly on culture suspension cells. Maximum total cell, CFC and CAFC production was seen in the first 4 weeks with up to 80-fold expansion in cell count, up to 4-fold expansion in CFC and up to 13-fold expansion in CAFC. Thereafter a continuous decrease in production of cells, CFC 2ndry CAFC was observed and cultures terminated at week 8. Mean telomere length of input PB CD34+ cells was 9,5 ± 0,5 kbp. After 4 weeks in culture, telomere length remained stable (9,6 ± 0,5 kbp). In 3/6 cultures terminated, cultures showed only a slightly decrease of telomere length compared to the input population (0,55 ± 0,1 kbp loss). However, in 2/6 cultures we could demonstrate an elevation of telomeres (+ 0,3 kbp) independent of a rapid loss of telomerase activity in all cultures during the culture period. Furthermore, the elevation of telomeres did not correlate with an enhanced stem/progenitor cell activity. These data confirm earlier results of granulocyte telomere change in myeloma patients following chemotherapy and tandem transplantation, where 154/193 patients showed an expected loss of telomeres during the treatment period, whereas 39/193 patients had an unexpected elevation of telomeres. We could now show in healthy donors that this phenomenon is independent of bone marrow stress due to chemotherapeutic treatment. In conclusion, we could show that the stromal coculture system with OP9/Tpo is highly effective in stem/progenitor cell expansion not only in CB but also in PB CD34+ cells. It is the first culture system, which allows expansion of hematpoietic cells without significant telomere erosion. We furthermore describe for the first time an age-independent healthy donor population which shows telomerase-independent, significant telomere elevation. Further studies have to demonstrate, if this phenomenon is potentially linked to a higher susceptibility for cancer disease.

scholarly journals Cell Source-Dependent In Vitro Chondrogenic Differentiation Potential of Mesenchymal Stem Cell Established from Bone Marrow and Synovial Fluid of Camelus dromedarius

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1918
Author(s):  
Young-Bum Son ◽  
Yeon Ik Jeong ◽  
Yeon Woo Jeong ◽  
Mohammad Shamim Hossein ◽  
Per Olof Olsson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Synovial Fluid ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Chondrocyte Differentiation ◽  
Differentiation Potential ◽  
Proliferation Capacity

Mesenchymal stem cells (MSCs) are promising multipotent cells with applications for cartilage tissue regeneration in stem cell-based therapies. In cartilage regeneration, both bone marrow (BM-MSCs) and synovial fluid (SF-MSCs) are valuable sources. However, the cellular characteristics and chondrocyte differentiation potential were not reported in either of the camel stem cells. The in vitro chondrocyte differentiation competence of MSCs, from (BM and SF) sources of the same Camelus dromedaries (camel) donor, was determined. Both MSCs were evaluated on pluripotent markers and proliferation capacity. After passage three, both MSCs showed fibroblast-like morphology. The proliferation capacity was significantly increased in SF-MSCs compared to BM-MSCs. Furthermore, SF-MSCs showed an enhanced expression of transcription factors than BM-MSCs. SF-MSCs exhibited lower differentiation potential toward adipocytes than BM-MSCs. However, the osteoblast differentiation potential was similar in MSCs from both sources. Chondrogenic pellets obtained from SF-MSCs revealed higher levels of chondrocyte-specific markers than those from BM-MSCs. Additionally, glycosaminoglycan (GAG) content was elevated in SF-MSCs related to BM-MSCs. This is, to our knowledge, the first study to establish BM-MSCs and SF-MSCs from the same donor and to demonstrate in vitro differentiation potential into chondrocytes in camels.

scholarly journals Bone Marrow Cell Aggregates: A Way of Collecting the Adult Human Hematopoietic Stem Cell Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4363-4363
Author(s):  
Alexandre Janel ◽  
Nathalie Boiret-Dupré ◽  
Juliette Berger ◽  
Céline Bourgne ◽  
Richard Lemal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Confocal Microscopy ◽  
Hematopoietic Stem Cell ◽  
Biological Samples ◽  
Mesenchymal Cells ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
Cd34 Cells

Abstract Hematopoietic stem cell (HSC) function is critical in maintaining hematopoiesis continuously throughout the lifespan of an organism and any change in their ability to self-renew and/or to differentiate into blood cell lineages induces severe diseases. Postnatally, HSC are mainly located in bone marrow where their stem cell fate is regulated through a complex network of local influences, thought to be concentrated in the bone marrow (BM) niche. Despite more than 30 years of research, the precise location of the HSC niche in human BM remains unclear because most observations were obtained from mice models. BM harvesting collects macroscopic coherent tissue aggregates in a cell suspension variably diluted with blood. The qualitative interest of these tissue aggregates, termed hematons, was already reported (first by I. Blaszek's group (Blaszek et al., 1988, 1990) and by our group (Boiret et al., 2003)) yet they remain largely unknown. Should hematons really be seen as elementary BM units, they must accommodate hematopoietic niches and must be a complete ex vivo surrogate of BM tissue. In this study, we analyzed hematons as single tissue structures. Biological samples were collected from i) healthy donor bone marrow (n= 8); ii) either biological samples collected for routine analysis by selecting bone marrow with normal analysis results (n=5); or iii) from spongy bone collected from the femoral head during hip arthroplasty (n=4). After isolation of hematons, we worked at single level, we used immunohistochemistry techniques, scanning electronic microscopy, confocal microscopy, flow cytometry and cell culture. Each hematon constitutes a miniature BM structure organized in lobular form around the vascular tree. Hematons are organized structures, supported by a network of cells with numerous cytoplasmic expansions associated with an amorphous structure corresponding to the extracellular matrix. Most of the adipocytes are located on the periphery, and hematopoietic cells can be observed as retained within the mesenchymal network. Although there is a degree of inter-donor variability in the cellular contents of hematons (on average 73 +/- 10 x103 cells per hematon), we observed precursors of all cell lines in each structure. We detected a higher frequency of CD34+ cells than in filtered bone marrow, representing on average 3% and 1% respectively (p<0.01). Also, each hematon contains CFU-GM, BFU-E, CFU-Mk and CFU-F cells. Mesenchymal cells are located mainly on the periphery and seem to participate in supporting the structure. The majority of mesenchymal cells isolated from hematons (21/24) sustain in vitro hematopoiesis. Interestingly, more than 90% of the hematons studied contained LTC-ICs. Furthermore, when studied using confocal microscopy, a co-localization of CD34+ cells with STRO1+ mesenchymal cells was frequently observed (75% under 10 µm of the nearest STRO-1+ cell, association statistically highly significant; p <1.10-16). These results indicate the presence of one or several stem cell niches housing highly primitive progenitor cells. We are confirming these in vitro data with an in vivo xenotransplantation model. These structures represent the elementary functional units of adult hematopoietic tissue and are a particularly attractive model for studying homeostasis of the BM niche and the pathological changes occurring during disease. Disclosures No relevant conflicts of interest to declare.

Epigenetic features in regulation of telomeres and telomerase in stem cells

2021 ◽  
Author(s):  
Fatma Dogan ◽  
Nicholas R. Forsyth
Keyword(s):  
Stem Cells ◽  
Telomere Length ◽  
Cell Biology ◽  
Replicative Senescence ◽  
Telomerase Activity ◽  
Cell Phenotype ◽  
Tissue Degeneration ◽  
Unique System ◽  
Telomerase Regulation

The epigenetic nature of telomeres is still controversial and different human cell lines might show diverse histone marks at telomeres. Epigenetic modifications regulate telomere length and telomerase activity that influence telomere structure and maintenance. Telomerase is responsible for telomere elongation and maintenance and is minimally composed of the catalytic protein component, telomerase reverse transcriptase (TERT) and template forming RNA component, telomerase RNA (TERC). TERT promoter mutations may underpin some telomerase activation but regulation of the gene is not completely understood due to the complex interplay of epigenetic, transcriptional, and posttranscriptional modifications. Pluripotent stem cells (PSCs) can maintain an indefinite, immortal, proliferation potential through their endogenous telomerase activity, maintenance of telomere length, and a bypass of replicative senescence in vitro. Differentiation of PSCs results in silencing of the TERT gene and an overall reversion to a mortal, somatic cell phenotype. The precise mechanisms for this controlled transcriptional silencing are complex. Promoter methylation has been suggested to be associated with epigenetic control of telomerase regulation which presents an important prospect for understanding cancer and stem cell biology. Control of down-regulation of telomerase during differentiation of PSCs provides a convenient model for the study of its endogenous regulation. Telomerase reactivation has the potential to reverse tissue degeneration, drive repair, and form a component of future tissue engineering strategies. Taken together it becomes clear that PSCs provide a unique system to understand telomerase regulation fully and drive this knowledge forward into aging and therapeutic application.

scholarly journals Melatonin Reverses the Loss of Stemness Induced by TNF-α in Human Bone Marrow Mesenchymal Stem Cells through Upregulation of YAP Expression

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Xudong Wang ◽  
Tongzhou Liang ◽  
Jincheng Qiu ◽  
Xianjian Qiu ◽  
Bo Gao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Molecular Mechanisms ◽  
Inflammatory Factor ◽  
Cell Transplant ◽  
Alizarin Red ◽  
Marrow Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration and disease treatment. However, long-term in vitro culture results in loss of MSC stemness. The inflammation that occurs at stem cell transplant sites (such as that resulting from TNF-α) is a contributing factor for stem cell treatment failure. Currently, there is little evidence regarding the protective role of melatonin with regard to the negative effects of TNF-α on the stemness of MSCs. In this study, we report a melatonin-based method to reduce the inflammatory effects on the stemness of bone marrow mesenchymal stem cells (BMMSCs). The results of colony formation assays, Alizarin red staining, western blotting, and reverse transcription-polymerase chain reactions suggest that melatonin can reverse the inflammatory damage caused by TNF-α treatment in the third, seventh, and tenth generations of primary BMMSCs (vs. control and the TNF-α-treated group). Meanwhile, a detailed analysis of the molecular mechanisms showed that the melatonin receptor and YAP signaling pathway are closely related to the role that melatonin plays in negative inflammatory effects against BMMSCs. In addition, in vivo experiments showed that melatonin could reverse the damage caused by TNF-α on bone regeneration by BMMSCs in nude mice. Overall, our results suggest that melatonin can reverse the loss of stemness caused by inflammatory factor TNF-α in BMMSCs. Our results also provide a practical strategy for the application of BMMSCs in tissue engineering and cell therapy.

scholarly journals Dose-Independent Therapeutic Benefit of Bone Marrow Stem Cell Transplantation after MI in Mice

Biomedicines ◽  
2020 ◽  
Vol 8 (6) ◽  
pp. 157
Author(s):  
Nicole Zarniko ◽  
Anna Skorska ◽  
Gustav Steinhoff ◽  
Robert David ◽  
Ralf Gaebel
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Ex Vivo ◽  
Therapeutic Benefit ◽  
Dependent Manner ◽  
Mesenchymal Progenitors

Several cell populations derived from bone marrow (BM) have been shown to possess cardiac regenerative potential. Among these are freshly isolated CD133+ hematopoietic as well as culture-expanded mesenchymal stem cells. Alternatively, by purifying CD271+ cells from BM, mesenchymal progenitors can be enriched without an ex vivo cultivation. With regard to the limited available number of freshly isolated BM-derived stem cells, the effect of the dosage on the therapeutic efficiency is of particular interest. Therefore, in the present pre-clinical study, we investigated human BM-derived CD133+ and CD271+ stem cells for their cardiac regenerative potential three weeks post-myocardial infarction (MI) in a dose-dependent manner. The improvement of the hemodynamic function as well as cardiac remodeling showed no therapeutic difference after the transplantation of both 100,000 and 500,000 stem cells. Therefore, beneficial stem cell transplantation post-MI is widely independent of the cell dose and detrimental stem cell amplification in vitro can likely be avoided.

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