scholarly journals Decidualization Potency and Epigenetic Changes in Human Endometrial Origin Stem Cells During Propagation

2021 ◽  
Vol 9 ◽  
Author(s):  
Elvina Valatkaitė ◽  
Raminta Baušytė ◽  
Aida Vitkevičienė ◽  
Diana Ramašauskaitė ◽  
Rūta Navakauskienė
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Reproductive System ◽  
Cell Cycle Control ◽  
Menstrual Blood ◽  
Great Promise ◽  
Epigenetic Changes ◽  
Similar Expression ◽  
Gene And Protein Expression

Human endometrium derived mesenchymal stem cells (hEndSCs) offer a great promise for regenerative medicine and reproductive system disorders treatment methods based on cell therapy due to their broad differentiation potential and highly efficient proliferation. In our study, we investigated the characteristics of hEndSCs that were isolated from two sources: endometrium and menstrual blood, which both contain endometrial origin stem cells. Changes in gene and protein expression levels during long-term cultivation and decidualization potential were examined in endometrial stem cells (EndSCs) and menstrual blood stem cells (MenSCs). The decidualization process was induced on early and late passages of hEndSCs using dibutyryl cyclic-AMP (db-cAMP) and medroxyprogesterone acetate (MPA) agents. We demonstrated that after long-term cultivation of hEndSCs the expression of typical mesenchymal stromal cell surface markers such as CD44, CD73, CD90, CD105 and perivascular marker CD146 remains at a similar level throughout long-term cultivation. Additionally, hematopoietic and endothelial markers CD34, CD45 were also tested, they were negative in all cases. Analyzed stem cells gene markers, such as OCT4, SOX2, NANOG, KLF4, showed similar expression in all passages of hEndSCs. RT-qPCR results demonstrated that the expression of cell cycle control associated genes - CDK2, CCNA2, CCNE2, p21, p53 and Rb, among all groups was very similar. Expression of genes associated with senescence (ATM, JUND, TOP2A, MYC) was maintained at a similar level throughout passaging. In addition, Western blot analysis was used to assess changes in proteins’ levels associated to epigenetics (EZH2, SUZ12, H3K27me3) and cell cycle control (cyclinE1, p53) during long-term cultivation. The levels of proteins associated with epigenetic changes were fluctuated slightly depending on the patient. Also, we demonstrated that in all induced hEndSCs the expression of decidualization markers Prolactin (PRL), IGFBP1 and WNT4 was upregulated. In conclusion, we demonstrated successful decidualization of stem cells derived from two reproductive system resources: endometrium and menstrual blood by using db-cAMP and MPA regardless of the length of the stem cell passaging. According these findings, we suppose that endometrium derived stem cells and menstrual blood derived stem cells could have a potency not only for endometrium tissue regeneration, but could also become a successful therapy for reproductive system disorders, including infertility or recurrent pregnancy loss.

scholarly journals DNA Methylation Changes duringIn VitroPropagation of Human Mesenchymal Stem Cells: Implications for Their Genomic Stability?

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Angela Bentivegna ◽  
Mariarosaria Miloso ◽  
Gabriele Riva ◽  
Dana Foudah ◽  
Valentina Butta ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Genomic Stability ◽  
Great Promise ◽  
Human Mscs ◽  
Low Oxygen ◽  
Long Term Culture ◽  
Functional Changes

Mesenchymal stem cells (MSCs) hold great promise for the treatment of numerous diseases. A major problem for MSC therapeutic use is represented by the very low amount of MSCs which can be isolated from different tissues; thusex vivoexpansion is indispensable. Long-term culture, however, is associated with extensive morphological and functional changes of MSCs. In addition, the concern that they may accumulate stochastic mutations which lead the risk of malignant transformation still remains. Overall, the genome of human MSCs (hMSCs) appears to be apparently stable throughout culture, though transient clonal aneuploidies have been detected. Particular attention should be given to the use of low-oxygen environment in order to increase the proliferative capacity of hMSCs, since data on the effect of hypoxic culture conditions on genomic stability are few and contradictory. Furthermore, specific and reproducible epigenetic changes were acquired by hMSCs duringex vivoexpansion, which may be connected and trigger all the biological changes observed. In this review we address current issues on long-term culture of hMSCs with a 360-degree view, starting from the genomic profiles and back, looking for an epigenetic interpretation of their genetic stability.

scholarly journals METTL3-mediated M6a Modification Regulates Cell Cycle Progression of Dental Pulp Stem Cells

2021 ◽  
Author(s):  
Haiyun Luo ◽  
Wenjing Liu ◽  
Yanli Zhang ◽  
Xiao Jiang ◽  
Shiqing Wu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tissue Engineering ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Differentially Expressed Genes ◽  
Dental Pulp ◽  
Cell Cycle Control ◽  
Dental Pulp Stem Cells ◽  
Differentially Expressed ◽  
Expression Level

Abstract Background: Dental pulp stem cells (DPSCs) exhibited self-renewal, pluripotency capacity and served as promising cells source in endodontic regeneration and tissue engineering. Meanwhile, the regenerative capacity of DPSCs is limited and reduced in long lifespan. N6-methyladenosine (m6A) is the most prevalent, reversible internal modification in RNAs. The methyltransferases complex and demethylases mediated m6A methylation and cooperated to impact various biological processes associated with stem cell fate determination. However, the biological effect of m6A methylation in DPSCs remained unclear. Methods: Cell surface markers and differentiation potential of primary DPSCs were identified and m6A immunoprecipitation with deep sequencing (m6A RIP-seq) was used to uncover characteristics of m6A modifications in DPSCs transcriptome. Expression level of m6A-related genes were evaluated in immature/mature pulp tissues and cells. Lentiviral vectors were constructed to knockdown or overexpress methyltransferase like 3 (METTL3). Cell morphology, viability, senescence and apoptosis were further analyzed by β-galactosidase, TUNEL staining and flow cytometry. Bioinformatic analysis combing m6A RIP and shMETTL3 RNA-seq was used to functionally enrich overlapped genes and screen target of METTL3. Cell cycle distributions were assayed by flow cytometry and m6A RIP-qPCR was used to confirm METTL3 mediated m6A methylation in DPSCs. Results: Here, m6A peaks distribution, binding area and motif in DPSCs were first revealed by m6A RIP-seq. We also found a relative high expression level of METTL3 in immature DPSCs with superior regenerative potential and METTL3 knockdown induced cell apoptosis and senescence. Furthermore, Conjoint analysis of m6A RIP and RNA-sequencing showed differentially expressed genes affected by METTL3 depletion was mainly enriched in cell cycle, mitosis and alteration of METTL3 expression resulted in cell cycle arrest which indicated METTL3 make essential effect in cell cycle control. To further investigate underlying mechanisms, we explored proteins interaction network of differentially expressed genes and Polo-like Kinase 1 (PLK1), a critical cycle modulator was identified as target of METTL3-mediated m6A methylation in DPSCs. Conclusions: These results revealed m6A methylated hallmarks in DPSCs and a regulatory role of METTL3 in cell cycle control. Our study shed light on therapeutic approaches in vital pulp therapy and serve new insight in stem cells based tissue engineering.

Role of cell cycle control in radiosensitization of mouse spermatogonial stem cells

2001 ◽  
Vol 77 (3) ◽  
pp. 357-363 ◽  
Author(s):  
P. P. W. Van Buul ◽  
A. Van Duyn-Goedhart ◽  
T. Beumer ◽  
A. L. Bootsma
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Cycle Control ◽  
Spermatogonial Stem Cells

Malignant Myeloma Cells Impair Phenotype and Function of stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1799-1799
Author(s):  
Ingmar Bruns ◽  
Sebastian Büst ◽  
Akos G. Czibere ◽  
Ron-Patrick Cadeddu ◽  
Ines Brückmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Plasma Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
Stem And Progenitor Cells

Abstract Abstract 1799 Poster Board I-825 Multiple myeloma (MM) patients often present with anemia at the time of initial diagnosis. This has so far only attributed to a physically marrow suppression by the invading malignant plasma cells and the overexpression of Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by malignant plasma cells triggering the death of immature erythroblasts. Still the impact of MM on hematopoietic stem cells and their niches is scarcely established. In this study we analyzed highly purified CD34+ hematopoietic stem and progenitor cell subsets from the bone marrow of newly diagnosed MM patients in comparison to normal donors. Quantitative flowcytometric analyses revealed a significant reduction of the megakaryocyte-erythrocyte progenitor (MEP) proportion in MM patients, whereas the percentage of granulocyte-macrophage progenitors (GMP) was significantly increased. Proportions of hematopoietic stem cells (HSC) and myeloid progenitors (CMP) were not significantly altered. We then asked if this is also reflected by clonogenic assays and found a significantly decreased percentage of erythroid precursors (BFU-E and CFU-E). Using Affymetrix HU133 2.0 gene arrays, we compared the gene expression signatures of stem cells and progenitor subsets in MM patients and healthy donors. The most striking findings so far reflect reduced adhesive and migratory potential, impaired self-renewal capacity and disturbed B-cell development in HSC whereas the MEP expression profile reflects decreased in cell cycle activity and enhanced apoptosis. In line we found a decreased expression of the adhesion molecule CD44 and a reduced actin polymerization in MM HSC by immunofluorescence analysis. Accordingly, in vitro adhesion and transwell migration assays showed reduced adhesive and migratory capacities. The impaired self-renewal capacity of MM HSC was functionally corroborated by a significantly decreased long-term culture initiating cell (LTC-IC) frequency in long term culture assays. Cell cycle analyses revealed a significantly larger proportion of MM MEP in G0-phase of the cell cycle. Furthermore, the proportion of apoptotic cells in MM MEP determined by the content of cleaved caspase 3 was increased as compared to MEP from healthy donors. Taken together, our findings indicate an impact of MM on the molecular phenotype and functional properties of stem and progenitor cells. Anemia in MM seems at least partially to originate already at the stem and progenitor level. Disclosures Off Label Use: AML with multikinase inhibitor sorafenib, which is approved by EMEA + FDA for renal cell carcinoma.

Cycling Marrow Stem Cells Are Lost with Purification.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2308-2308
Author(s):  
Laura R Goldberg ◽  
Mark S Dooner ◽  
Mandy Pereira ◽  
Michael DelTatto ◽  
Elaine Papa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tritiated Thymidine ◽  
Hematopoietic Stem ◽  
Cell Purification ◽  
Marrow Stem Cell ◽  
Marrow Cells

Abstract Abstract 2308 Hematopoietic stem cell biologists have amassed a tremendous depth of knowledge about the biology of the marrow stem cell over the past few decades, facilitating invaluable basic scientific and translational advances in the field. Most of the studies to date have focused on highly purified populations of marrow cells, with emphasis placed on the need to isolate increasingly restricted subsets of marrow cells within the larger population of resident bone marrow cells in order to get an accurate picture of the true stem cell phenotype. Such studies have led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to homogeneity. However, work from our laboratory, focusing on the stem cell potential in un-separated whole bone marrow (WBM), supports an alternate view of marrow stem cell biology in which a large population of marrow stem cells are actively cycling, continually changing phenotype with cell cycle transit, and therefore, cannot be purified to homogeneity. Our studies separating WBM into cell cycle-specific fractions using Hoechst 33342/Pyronin Y or exposing WBM to tritiated thymidine suicide followed by competitive engraftment into lethally irradiated mice revealed that over 50% of the long-term multi-lineage engraftment potential in un-separated marrow was due to cells in S/G2/M. This is in stark contrast to studies showing that highly purified stem cell populations such as LT-HSC (Lineage–c-kit+sca-1+flk2−) engraft predominantly when in G0. Additionally, by performing standard isolation of a highly purified population of stem cells, SLAM cells (Lineage–c-kit+sca-1+flk2−CD150+CD41−CD48−), and testing the engraftment potential of different cellular fractions created and routinely discarded during this purification process, we found that 90% of the potential engraftment capacity in WBM was lost during conventional SLAM cell purification. Incubation of the Lineage-positive and Lineage-negative fractions with tritiated thymidine, a DNA analogue which selectively kills cells traversing S-phase, led to dramatic reductions in long-term multi-lineage engraftment potential found within both cellular fractions (over 95% and 85% reduction, respectively). This indicates that the discarded population of stem cells during antibody-based stem cell purification is composed largely of cycling cells. In sum, these data strongly support that 1) whole bone marrow contains actively cycling stem cells capable of long-term multi-lineage engraftment, 2) these actively cycling marrow stem cells are lost during the standard stem cell purification strategies, and 3) the protean phenotype of actively cycling cells as they transit through cell cycle will render cycling marrow stem cells difficult to purify to homogeneity. Given the loss of a large pool of actively cycling HSC during standard stem cell isolation techniques, these data underscore the need to re-evaluate the total hematopoietic stem cell pool on a population level in addition to a clonal level in order to provide a more comprehensive study of HSC biology. Disclosures: No relevant conflicts of interest to declare.

Long Term Maintenance of Myeloid Leukemia Stem Cell-Like Populations Cultured with Mesenchymal Stromal Cells (MSC)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3546-3546
Author(s):  
Sawa Ito ◽  
A. John Barrett ◽  
Andre Larochelle ◽  
Nancy F. Hensel ◽  
Keyvan Keyvanfar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Culture Conditions ◽  
Myelogenous Leukemia ◽  
Cell Cycle Analysis ◽  
Remission Induction ◽  
Leukemia Stem Cell

Abstract Abstract 3546 Because MSC support the growth and the differentiation of normal hematopoietic stem cells we hypothesized that MSC might also support leukemia cells, in particular leukemia stem cells (LSC) in vitro. We cultured blast cells from patients with acute myelogenous leukemia (AML) in liquid medium to study persistence of stem-cell-like and differentiated leukemia cell populations by flow cytometry, with and without MSC and additional growth factors. Cryopresrerved peripheral blood mononuclear cells (PBMC) were obtained from 6 AML patients (mean Age 47, range 23–74). Leukemia blasts were isolated by sorting live (propidium iodide (PI)-negative) CD34+ lineage (CD2+, CD3+, CD14+ and CD19+) -negative cells using a FACS ARIA II cell sorter (BD). Sorted blasts (2.5 ×105 cells) were co-cultured with an equal number of irradiated MSC derived from healthy donor bone marrow in RPMI medium supplemented with 10% human serum, with or without a human cytokine (CYTO) mixture (50 ng/ml interleukin 3, 150 ng/ml stem cell factor, and 150ng/ml Flt-3 ligand). MSC were replenished every two weeks. The phenotype of cultured cells was analyzed weekly using fluorescently-conjugated monoclonal antibodies against CD34, CD38, and CD45, plus the lineage panel and a dead cell exclusion dye Cell cycle analysis with Hoeschst 33342 and Pyronin Y was performed on cells co-stained with CD34, CD45 and PI. Primary leukemia samples were phenotypically heterogeneous with respect to proportions of cells (co-)staining for CD34 and CD38 as previously reported: three samples showed CD34+CD38- predominance (LSC-like leukemia), and three were CD34+CD38+ (common myeloid progenitor (CMP)-like leukemia). LSC-like leukemia maintained viable CD34+CD38- cells for at least 6 weeks when co-cultured with MSC alone, in contrast to cultures with cytokines or medium only which showed rapid decline in the LSC populations and no prolonged maintenance of viable cells (p=0.0005) (Figure, left panel). CMP-like leukemia maintained their CD34+CD38+ phenotype when co-cultured with MSC alone but persistence of this subset was not significantly different from the other culture conditions (p=0.5) and no culture remained viable after 4 weeks (Figure, right panel). Cell cycle analysis showed that co-culture with MSC maintained CD34+ blasts in G0 significantly more than other culture conditions (P<0.0001). We conclude that MSC support the maintenance of a leukemia stem cell phenotype in a long- term (6 week) in vitro culture system. The differential capacity of MSC to support LSC- like and CMP- like leukemia may be associated with the different frequency of leukemia initiating cells within each leukemic blast population. NSG mice xenotranplant model experiments are ongoing to confirm this hypothesis. Co-culture of LSC with MSC represents a simple approach to maintain LSC in vitro and could be utilized to screen the drug targeting LSCs. Further study of the effect of MSC on LSC would elucidate a potential mechanism whereby the marrow microenvironment serves as a reservoir of persisting leukemia after remission induction chemotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Safety of Mesenchymal Stem Cells for Clinical Application

2012 ◽  
Vol 2012 ◽  
pp. 1-4 ◽  
Author(s):  
Youwei Wang ◽  
Zhi-bo Han ◽  
Yong-ping Song ◽  
Zhong Chao Han
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Regenerative Medicine ◽  
Therapeutic Agents ◽  
Great Promise ◽  
Safety Issues ◽  
Human Use

Mesenchymal stem cells (MSCs) hold great promise as therapeutic agents in regenerative medicine and autoimmune diseases, based on their differentiation abilities and immunosuppressive properties. However, the therapeutic applications raise a series of questions about the safety of culture-expanded MSCs for human use. This paper summarized recent findings about safety issues of MSCs, in particular their genetic stability in long-termin vitroexpansion, their cryopreservation, banking, and the role of serum in the preparation of MSCs.

Cell Cycle Control of Embryonic Stem Cells

2005 ◽  
Vol 1 (2) ◽  
pp. 131-138 ◽  
Author(s):  
Josephine White ◽  
Stephen Dalton
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Cycle Control ◽  
Embryonic Stem

scholarly journals Hydroxyurea Can Be Used to Increase Mouse c-kit+Thy-1.1loLin−/loSca-1+ Hematopoietic Cell Number and Frequency in Cell Cycle In Vivo

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4354-4362 ◽  
Author(s):  
Nobuko Uchida ◽  
Annabelle M. Friera ◽  
Dongping He ◽  
Michael J. Reitsma ◽  
Ann S. Tsukamoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Cell Number ◽  
Short Term ◽  
Synthesis Inhibitor ◽  
Hematopoietic Stem ◽  
Cell Cycle Status

Abstract The DNA synthesis inhibitor hydroxyurea (HU) was administered to determine whether it induces changes in the cell-cycle status of primitive hematopoietic stem cells (HSCs)/progenitors. Administration of HU to mice leads to bone marrow accumulation of c-kit+Thy-1.1loLin−/loSca-1+ (KTLS) cells in S/G2/M phases of the cell cycle. HU is a relatively nontoxic, reversible cell-cycle agent that can lead to approximately a threefold expansion of KTLS cells in vivo and approximately an eightfold increase in the number of KTLS cells in S/G2/M. HSCs in HU-treated mice have undiminished multilineage long-term and short-term clonal reconstitution activity.

scholarly journals Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3704-3704
Author(s):  
Aldona A Karaczyn ◽  
Edward Jachimowicz ◽  
Jaspreet S Kohli ◽  
Pradeep Sathyanarayana
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Quiescent State ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

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