Cyclin C Regulates the Quiescence of Human CD34+CD38- Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1273-1273
Author(s):  
Yasuhiko Miyata ◽  
Yan Liu ◽  
Vladimir Jankovic ◽  
Goro Sashida ◽  
Silvia Menendez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fold Increase ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cyclin C

Abstract The relative quiescence of adult hematopoietic stem cells (HSCs) at steady state represents an important regulatory mechanism for maintaining their self-renewal and engraftment capacity, as well as their resistance to cytotoxic insults. However, the specific mechanisms regulating the intermittent entry of HSCs into the cell cycle are not well characterized. Here we provide the evidence that cyclin C (CCNC) specifically promotes the G0/G1 transition of human CD34+CD38- HSCs, and thus can significantly affect the loss of HSC self-renewal capacity in in vitro culture. Based on the recently hypothesized specific function of CCNC in G0 exit of human fibroblasts, we have analyzed the effects of CCNC loss on the behavior of human cord blood HSCs. We achieved a highly efficient knockdown of CCNC expression (>90%) using lentiviral shRNA (shCCNC) transduction of freshly isolated human cord blood CD34+ cells, allowing the in vitro assessment of early cell cycle regulation in HSCs. First, we observed a 3-fold increase in the G0 fraction of shCCNC transduced CD34+ cells compared to the empty vector control, based on the Pyronin Y and Hoechst 33342 staining 72h after infection. The depletion of CCNC did not prevent cell cycle progression beyond the G1 entry, as we observed no significant changes in the G1/S/G2-M distribution, indicating that critical CCNC activities may be restricted to the G0 checkpoint. Consistent with the reported enrichment of functional HSCs in the G0 fraction, CCNC knockdown (CCNC KD) cells showed increased activity in all surrogate in vitro assays of stem cell-ness tested: a ∼3 fold increase in CD34+ population after long term culture, a ∼2.5 fold increase in long-term culture initiating cells (LTC-ICs) and a ∼3.5 fold increase in cobblestone area forming cells (CAFCs). In contrast, CFU assays using freshly sorted shCCNC cells (and cells obtained after one-week culture in cytokines) showed only a minimal decrease in total colony number, with no difference in colony composition or morphology, indicating no significant effect on hematopoietic progenitor cell differentiation. However, we did observe a prominent effect on secondary CFUs after 2 and 3 weeks in liquid culture (i.e. using the delta assay), namely a 2-fold and 30-fold increase in shCCNC over control culture respectively, again indicating a specific function of CCNC on the more primitive cells. Consistently, CCNC KD robustly enhanced CD34 expression and secondary CFU maintenance in sorted CD34+CD38- cells (HSCs); both markers of hematopoietic cell immaturity were rapidly lost in CD34+CD38+ cells (i.e. the committed progenitor cells) with no detectable effect of shCCNC transduction. Finally, we have found that these effects of CCNC depletion are likely the result of its initial loss of function, as transient CCNC KD, using siRNA transfection of CD34+ cells, produced similar biological effects as the constitutive lentiviral shCCNC expression. Collectively, these data indicate a cell context-dependent effect of CCNC KD on the initial rate of cell cycle entry by quiescent HSCs and suggest that this approach could be used to preserve their functional capacity in culture, potentially enhancing the ex vivo expansion of HSCs, as well as their use in gene therapy protocols. Transplantation of transduced CD34+ cells into sublethally irradiated immunodeficient mice is now under way to establish the potentially beneficial effects of CCNC KD on the engraftment and repopulating capability of cultured HSCs.

Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2370-2370
Author(s):  
Daniel Ewerth ◽  
Stefanie Kreutmair ◽  
Birgit Kügelgen ◽  
Dagmar Wider ◽  
Julia Felthaus ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Research Funding ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Ex-Vivo Expansion of Human Cord Blood CD34+ Cells by Overexpression of Bmi-1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1329-1329
Author(s):  
Aleksandra Rizo ◽  
Edo Vellenga ◽  
Gerald de Haan ◽  
Jan Jacob Schuringa
Keyword(s):  
Cord Blood ◽  
Cell Expansion ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into cells of all hematopoietic lineages. Because of this unique property, they are used for HSC transplantations and could serve as a potential source of cells for future gene therapy. However, the difficulty to expand or even maintain HSCs ex vivo has been a major limitation for their clinical applications. Here, we report that overexpression of the Polycomb group gene Bmi-1 in human cord blood-derived HSCs can potentially overcome this limitation as stem/progenitor cells could be maintained in liquid culture conditions for over 16 weeks. In mouse studies, it has been reported that increased expression of Bmi-1 promotes HSC self-renewal, while loss-of-function analysis revealed that Bmi-1 is implicated in maintenance of the hematopoietic stem cells (HSC). In a clinically more relevant model, using human cord blood CD34+ cells, we have established a long-term ex-vivo expansion method by stable overexpression of the Bmi-1 gene. Bmi-1-transduced cells proliferated in liquid cultures supplemented with 20% serum, SCF, TPO, Flt3 ligand, IL3 and IL6 for more than 4 months, with a cumulative cell expansion of more then 2×105-fold. The cells remained cytokine-dependent, while about 4% continued to express CD34 for over 20 weeks of culture. The cultured cells retained their progenitor activity throughout the long-term expansion protocol. The colony-forming units (CFUs) were present at a frequency of ~ 30 colonies per 10 000 cells 16 weeks after culture and consisted of CFU-GM, BFU-E and high numbers of CFU-GEMM type progenitors. After plating the transduced cells in co-cultures with the stromal cell line MS5, Bmi-1 cells showed a proliferative advantage as compared to control cells, with a cumulative cell expansion of 44,9 fold. The non-adherent cells from the co-cultures gave rise to higher numbers of colonies of all types (~70 colonies/10.000 cells) after 4 weeks of co-culture. The LTC-IC frequencies were 5-fold higher in the Bmi-1-transduced cells compared to control cells (1/361 v.s. 1/2077, respectively). Further studies will be focused on in-vivo transplantation of the long-term cultured cells in NOD/SCID mice to test their repopulating capacity. In conclusion, our data implicate Bmi-1 as an important modulator of human HSC self-renewal and suggest that it can be a potential target for therapeutic manipulation of human HSCs.

Enforced Expression of GATA-2 Confers Quiescence on Human Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 83-83
Author(s):  
Alex J. Tipping ◽  
Cristina Pina ◽  
Anders Castor ◽  
Ann Atzberger ◽  
Dengli Hong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Zinc Finger ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Colony Number

Abstract Hematopoietic stem cells (HSCs) in adults are largely quiescent, periodically entering and exiting cell cycle to replenish the progenitor pool or to self-renew, without exhausting their number. Expression profiling of quiescent HSCs in our and other laboratories suggests that high expression of the zinc finger transcription factor GATA-2 correlates with quiescence. We show here that TGFβ1-induced quiescence of wild-type human cord blood CD34+ cells in vitro correlated with induction of endogenous GATA-2 expression. To directly test if GATA-2 has a causative role in HSC quiescence we constitutively expressed GATA-2 in human cord blood stem and progenitor cells using lentiviral vectors, and assessed the functional output from these cells. In both CD34+ and CD34+ CD38− populations, enforced GATA-2 expression conferred increased quiescence as assessed by Hoechst/Pyronin Y staining. CD34+ cells with enforced GATA-2 expression showed reductions in both colony number and size when assessed in multipotential CFC assays. In CFC assays conducted with more primitive CD34+ CD38− cells, colony number and size were also reduced, with myeloid and mixed colony number more reduced than erythroid colonies. Reduced CFC activity was not due to increased apoptosis, as judged by Annexin V staining of GATA-2-transduced CD34+ or CD34+ CD38− cells. To the contrary, in vitro cultures from GATA-2-transduced CD34+ CD38− cells showed increased protection from apoptosis. In vitro, proliferation of CD34+ CD38− cells was severely impaired by constitutive expression of GATA-2. Real-time PCR analysis showed no upregulation of classic cell cycle inhibitors such as p21, p57 or p16INK4A. However GATA-2 expression did cause repression of cyclin D3, EGR2, E2F4, ANGPT1 and C/EBPα. In stem cell assays, CD34+ CD38− cells constitutively expressing GATA-2 showed little or no LTC-IC activity. In xenografted NOD/SCID mice, transduced CD34+ CD38−cells expressing high levels of GATA-2 did not contribute to hematopoiesis, although cells expressing lower levels of GATA-2 did. This threshold effect is presumably due to DNA binding by GATA-2, as a zinc-finger deletion variant of GATA-2 shows contribution to hematopoiesis from cells irrespective of expression level. These NOD/SCID data suggest that levels of GATA-2 may play a part in the in vivo control of stem and progenitor cell proliferation. Taken together, our data demonstrate that GATA-2 enforces a transcriptional program on stem and progenitor cells which suppresses their responses to proliferative stimuli with the result that they remain quiescent in vitro and in vivo.

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.

STAT5-Induced Self-Renewal and Impaired Myelopoiesis of Human Hematopoietic Stem/Progenitor Cells Involves Downmodulation of C/EBPα.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 268-268
Author(s):  
Jan Jacob Schuringa ◽  
Bart-Jan Wierenga ◽  
Hein Schepers ◽  
Malcolm A.S. Moore ◽  
Edo Vellenga
Keyword(s):  
Progenitor Cells ◽  
Rt Pcr ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Affymetrix Microarray ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Cd34 Cells ◽  
Proliferative Advantage

Abstract Previously, we demonstrated that enforced activation of STAT5 in human cord blood (CB)-derived stem/progenitor cells results in enhanced long-term stem cell self-renewal and impaired myelopoiesis (J.J.Schuringa et al, J.Exp.Med. 2004;200:623). Now, C/EBPα was identified as a critical transcription factor that is downregulated by STAT5. Affymetrix microarray analysis on STAT5A(1*6)-transduced CD34+ cells identified C/EBPα as the most prominently downregulated gene (−3.3 fold), and these data were confirmed by RT-PCR and Western blotting. To determine the cell-biological relevance of these observations, a 4-OHT-inducible C/EBPα-ER protein was co-expressed with the STAT5A(1*6) mutant in CB CD34+ cells by using a retroviral approach. Re-expression of C/EBPα in STAT5A(1*6) cells resulted in a marked restoration of myelopoiesis as determined by morphological analyses, FACS analyses for myeloid markers such as CD11b, CD14 and CD15, and RT-PCR for myeloid-restricted genes such as g-csfr. While enforced activation of STAT5A resulted in accelerated erythropoiesis, this was blocked when C/EBPα was re-introduced into STAT5A(1*6) cells. Similarly, the proliferative advantage imposed on CD34+ cells by STAT5A(1*6) depended on the downmodulation of C/EBP as reintroduction of C/EBPα in these cells induced a quick cell cycle arrest and the onset of myeloid differentiation. At the stem/progenitor cell level, LTC-IC frequencies were elevated from 0.5% to 11% by STAT5A(1*6) as compared to controls, but these elevated LTC-IC frequencies were strongly reduced when C/EBPα was reintroduced in STAT5A(1*6) cells. Enumeration of progenitors in methylcellulose assays revealed similar results, the number of CFCs was reduced over 10-fold when C/EBPα was expressed in STAT5A(1*6) cells. Also, secondary CAFCs and long-term cultures could only be generated from STAT5A(1*6) expressing cells, but not from cells that co-expressed STAT5A(1*6) and C/EBPα. Taken together, these data indicate that STAT5-induced self-renewal and impaired myelopoiesis involves downmodulation of C/EBPα.

Identification of a Hierarchy of Multipotent Hematopoietic Progenitors in Human Cord Blood.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2237-2237
Author(s):  
Ravindra Majeti ◽  
Christopher Y. Park ◽  
Irving L. Weissman
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Abstract Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC have been identified, as have downstream lineage-committed progenitors, but not multipotent progenitors. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow, and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. While, the function of the CD90- subpopulations is unknown, the CD90+CD45RA- subpopulation presumably contains HSC. We report here in vitro and in vivo functional studies of these three subpopulations from normal human cord blood. In vitro, CD90+CD45RA- cells formed all types of myeloid colonies in methylcellulose and were able to replate with 70% efficiency. CD90-CD45RA- cells also formed all types of myeloid colonies, but replated with only 33% efficiency. CD90-CD45RA+ cells failed to form myeloid colonies in methylcellulose. In liquid culture, CD90+CD45RA- cells gave rise to all three subpopulations; CD90-CD45RA- cells gave rise to both CD90- subpopulations, but not CD90+ cells; CD90-CD45RA+ cells gave rise to themselves only. These data establish an in vitro differentiation hierarchy from CD90+CD45RA- to CD90-CD45RA- to CD90-CD45RA+ cells among Lin-CD34+CD38- cord blood. In vivo, xenotransplantation of CD90+CD45RA- cells into NOD/SCID/IL-2R?-null newborn mice resulted in long-term multilineage engraftment with transplantation of as few as 10 purified cells. Secondary transplants from primary engrafted mice also resulted in long-term multilineage engraftment, indicating the presence of self-renewing HSC. Transplantation of CD90-CD45RA- cells also resulted in long-term multilineage engraftment; however, secondary transplants did not reliably result in long-term engraftment, indicating a reduced capacity for self-renewal. Transplantation of CD90-CD45RA+ cells did not result in any detectable human hematopoietic cells, indicating that the function of these cells is undetermined. Finally, transplantation of limiting numbers of CD90-CD45RA- cells (less than 100) resulted in multilineage human engraftment at 4 weeks, that was no longer detectable by 12 weeks. Thus, the CD90-CD45RA- subpopulation is capable of multilineage differentiation while exhibiting limited self-renewal ability. We believe this study represents the first prospective identification of a population of human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.

A Short Pulse of Prostaglandin E2 (PGE2) Induces Long Term Chromatin Changes in Hematopoietic Stem Cells Leading to Increased Self-Renewal and Engraftment

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 246-246
Author(s):  
Eva M Fast ◽  
Ellen M Durand ◽  
Audrey Sporrij ◽  
Leslie Ojeaburu ◽  
Rebecca Maher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Open Chromatin ◽  
Advisory Committees ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Induced Genes

Abstract Hematopoietic stem cells (HSCs) offer promising treatment options for many blood diseases. We have previously identified Prostaglandin E2 (PGE2), a small molecule that increased HSC numbers in the zebrafish embryo. In an adult mammalian transplantation setting a two hour treatment significantly enhanced HSC engraftment. Currently PGE2 is being tested in a phase 2 clinical trial to improve cord blood transplants. To better understand PGE2 effect on HSCs mouse multipotent progenitors (MPP), short term (ST) HSCs, and long term (LT) HSCs were isolated via FACS and given a two hour pulse of PGE2 followed by a competitive transplantation assay. Surprisingly, PGE2 treatment mainly affected ST-HSCs by dramatically prolonging their ability to contribute to peripheral blood. The effect of the two hour treatment persisted through secondary competitive transplants in which robust peripheral blood chimerism of ST-HSCs was evident even 1.5 years after having been exposed to the drug. To elucidate underlying molecular changes gene expression right after PGE2 treatment as well as in ST-HSCs after transplantation was assessed. PGE2 target genes were divided into two categories; "transiently induced" and "permanently induced" genes. Most of the transcripts upregulated two hour after PGE2 treatment were "transiently induced" meaning that they did not continue to be differentially expressed after transplantation. In contrast, a few transcripts including chemokines such as Cxcl2, Cxcl3, members of the Fos gene family as well as Nr4a1, 2 and 3 were both upregulated right after PGE2 treatment as well as in ST-HSCs after transplantation. We classified these genes as "permanently induced". ATAC (Assay for Transposase-Accessible Chromatin)-seq analysis of the transplanted PGE2 treated cells indicated that these "permanently induced" genes maintained a distinctly open chromatin profile in both promotor and enhancer regions, whereas the "transiently induced" genes did not. Gene expression in human CD34+ cells included a signature implying CREB as the main transcription factor responsible for the acute PGE2 response. Phospho-FACS in mouse ST-HSCs and Western-blot analysis in human CD34+ cells confirmed a significant increase in CREB phosphorylation after PGE2 stimulation. Chromatin immunoprecipitation (ChIP)-seq analysis of pCREB was able to identify specific genomic regions where pCREB is recruited to after PGE2 treatment. Compared to unstimulated CD34+ cells an increased binding of pCREB could be detected in promotor regions near transcription start sites. In addition over 90% of de-novo pCREB binding occurred in intergenic and intronic regions. To determine the activation state of these putative enhancers changes in the histone mark H3K27ac and open chromatin state (via ATAC-seq) were assessed after PGE2 treatment. The data suggest that PGE2-induced pCREB binding correlates with remodeling of chromatin already after two hours of drug treatment. Furthermore chromatin sites opened by PGE2 were significantly enriched for the CREB motif both in human CD34+ cells acutely after treatment as well as in mouse ST-HSCs 1.5 years after transplant. In summary this work shows that a two hour treatment with PGE2 is sufficient to confer long-term engraftment properties to ST-HSCs. PGE triggers a chromatin remodeling event through CREB that can permanently alter epigenetic state and gene expression of ST-HSCs. Understanding the self-renewal network induced by PGE2 will not only enrich current clinical applications targeted at increasing engraftable HSC numbers but also further basic understanding of HSC self-renewal. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Interferon-γ Perturbs Key Signaling Pathways Induced By Thrombopoietin, but Not Eltrombopag, in Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3870-3870 ◽  
Author(s):  
Hai Cheng ◽  
Patali S. Cheruku ◽  
Luigi Alvarado ◽  
Ayla Cash ◽  
Cynthia E. Dunbar ◽  
...  
Keyword(s):  
Cell Signaling ◽  
Signaling Pathways ◽  
Research Funding ◽  
Fold Increase ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Inflammatory Conditions ◽  
Cd34 Cells

Abstract Thrombopoietin (TPO) is the main regulator of hematopoietic stem and progenitor cell (HSPC) self-renewal and survival. Upon binding to its receptor, c-MPL, TPO activates cell signaling, through JAK-STAT and other pathways, which is tightly balanced by negative regulatory signaling processes. Recent studies indicate that chronic exposure of HSPCs to IFNγ, as exemplified in subjects with severe aplastic anemia (SAA), impairs self-renewal by perturbing TPO signaling pathways. Despite elevated levels of TPO in subjects with SAA, the TPO receptor agonist Eltrombopag (Epag) improves trilineage hematopoiesis in refractory SAA, suggesting that it may activate signaling within HSPC in a way that is distinct from TPO under inflammatory conditions. To address the paradox of Epag efficacy despite high endogenous TPO levels in bone marrow failure, G-CSF mobilized human CD34+ cells from 6 healthy donors were cultured in the presence of SCF, FLT3 and either TPO 5 ng/ml (TPO5) or Epag 3 μg/ml (Epag), with or without IFNγ 100 ng/ml. After 7 days in culture, cells were characterized via flow cytometry, CFU assay and transplantation in immunodeficient (NSG) mice. The percentages of CD34+ cells in cultures containing TPO5 or Epag alone were similar (83.3 ± 9.7% and 87.6 ± 7.1%, respectively), but were better preserved with Epag than TPO5 in the presence of IFNγ (46.7 ± 16.1% and 24.6 ± 15.0% respectively, p<0.05). Accordingly, when comparing 7-day cultures with and without IFNγ, the absolute numbers of CD34+ cells were markedly reduced with TPO5 (average 7.6-fold, p<0.005) but only minimally decreased with Epag (average 1.6-fold, p = n.s.). The adjusted numbers of CFUs after 7 days in the presence of IFNγ similarly decreased 2.7-fold with TPO5 but remained unchanged with Epag compared to cultures without IFNγ. When the 7-day expanded progeny of an equal starting number of CD34+ cells was transplanted in NSG mice, human cell engraftment was superior with Epag (34 ± 3.8% human CD45+ cells) than with TPO5 (21 ± 1.8% human CD45+ cells, p<0.05) cultures in the presence of IFNγ, suggesting an impact of Epag on the most primitive long-term repopulating HSPCs. To investigate potential mechanisms by which Epag positively affects maintenance of HSPCs under inflammatory conditions, we examined cell signaling pathways induced upon binding of TPO, Epag and IFNγ to their respective receptors in human CD34+ cells. At a concentration of 5ng/mL, TPO induced a rapid (peak < 1 hour) and high potency rise in STAT5 phosphorylation followed by a rapid (< 2 hours) decay in signal. In contrast, Epag induced a slow (peak 4 hours) low potency rise in STAT5 phosphorylation, and the signal persisted for at least 10 hours. The difference in cell signaling potency and kinetics between TPO and Epag is likely related to their binding to distinct regions of c-MPL, resulting in alternate receptor conformational changes. We next investigated the impact of IFNγ on TPO and Epag-induced STAT5 phosphorylation at the signal peak (<1 and 4 hours, respectively). As previously shown in murine HSPCs, IFNγ impaired TPO signaling in human HSPCs (Figure, panels A, C). In contrast, Epag-induced STAT5 phosphorylation was preserved or increased in the presence of IFNγ (Figure, panels B, C). When Epag and TPO were combined, inhibition of TPO signaling by IFNγ was partially restored (Figure, panel D). By reducing the dose of TPO from 5 to 1ng/mL, and therefore reducing the potency of signaling to levels similar to Epag, the inhibitory effect of IFNγ on TPO signaling was abolished (Figure, panel E). Activation of IFNγ receptor by its ligand induces phosphorylation of STAT1 and subsequent expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of both IFNγ and c-MPL receptors via inhibition of STAT1 and STAT5 phosphorylation, respectively. We found that IFNγ-induced phosphorylation of STAT1 was increased in the presence of TPO 5ng/mL (1.5-fold increase, p<0.05) but unaffected by Epag (1.1-fold increase, p = n.s.) or TPO 1ng/mL (1.1-fold increase, p = n.s.). Our data indicate that Epag counters IFNγ-induced perturbation of TPO signaling in human HSPCs. Epag produces an unopposed low potency, slow kinetic positive signal and activates c-Mpl above a threshold level critical for HSPC self-renewal. Epag's evasion of IFN blockade of a critical pathway of growth factor cell signaling may explain its efficacy in improving hematopoiesis in SAA. Figure Figure. Disclosures Cheng: Novartis: Research Funding. Cheruku:Novartis: Research Funding. Alvarado:Novartis: Research Funding. Cash:Novartis: Research Funding. Dunbar:Novartis: Research Funding. Young:Novartis: Research Funding. Larochelle:Novartis: Research Funding.

The Role of Cyclin C on Hematopoietic Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4238-4238
Author(s):  
Yasuhiko Miyata ◽  
Yan Lie ◽  
Goro Sashida ◽  
Stephen Nimer
Keyword(s):  
Cell Cycle ◽  
Biological Effects ◽  
Human Fibroblasts ◽  
Hematopoietic Cells ◽  
Transduction Efficiency ◽  
Quiescent State ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cyclin C

Abstract The regulation of pluripotent hematopoietic stem cells (HSCs) is essential for the maintenance of multilineage blood production throughout life. The ability to self-renew or differentiate is a critical factor in the regulation of HSC numbers and cell fate, but so is the ability to enter and reside in a quiescent state (G0 phase of the cell cycle). The regulation of the G0 stem cell compartment is important for maintaining an inexhaustible pool of HSCs, protected from cellular stress. We have recently shown that the ETS transcription factor MEF is an important regulator of HSC quiescence (H. D. Lacorazza, Cancer Cell, 2006). The mechanism by which MEF or cell cycle regulatory proteins like p21 control entry into (and exit from) the G0 stage of the cell cycle is poorly understood. Insights into the mechanisms of HSC regulation could have important therapeutic potential. Recently, a Cyclin C/cdk3 interaction was reported to play an important role in cell cycle re-entry from quiescent state (G0 to G1) by phosphorylating the retinoblastoma protein (Rb) in human fibroblasts (S. Ren, Cell, 2004). To elucidate if cyclin C is involved in the regulation of cell cycle in hematopoietic cells, especially human CD34+ HSCs, we have optimized a FG12 lentivirus based shRNA strategy (kindly provided by D. Baltimore, PNAS, 2002). shRNA directed against cyclin C successfully decreased both cyclin C mRNA and protein expression levels. Using this vector, we have consistently achieved a 90 % transduction efficiency and a 60–70 % reduction of cyclin C levels in human CD34+ cells as evidenced by real time PCR. Now we are analyzing the cell cycle and biological effects of cyclin C knockdown and overexpression on hematopoietic cells using both human CD34+ cells and human leukemic cell lines.

STAT5-induced self-renewal and impaired myelopoiesis of human hematopoietic stem/progenitor cells involves down-modulation of C/EBPα

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4326-4333 ◽  
Author(s):  
Albertus T. J. Wierenga ◽  
Hein Schepers ◽  
Malcolm A. S. Moore ◽  
Edo Vellenga ◽  
Jan Jacob Schuringa
Keyword(s):  
Progenitor Cells ◽  
Pcr Analysis ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Cd34 Cells ◽  
Polymerase Chain ◽  
Proliferative Advantage

AbstractPreviously, we demonstrated that enforced activation of signal transducer and activator of transcription 5 (STAT5A) in human cord blood (CB)–derived stem/progenitor cells results in enhanced self-renewal and impaired myelopoiesis. The present study identifies C/EBPα as a critical component that is down-regulated by STAT5. Microarray and reverse transcriptase–polymerase chain reaction (RT-PCR) analysis on STAT5A1*6-transduced CD34+ cells identified C/EBPα as the most prominently down-regulated gene. To determine the cell-biological relevance of these observations, a 4-OHT-inducible C/EBPα-ER protein was co-expressed with the STAT5A1*6 mutant in CB CD34+ cells using a retroviral approach. Re-expression of C/EBPα in STAT5A1*6 cells resulted in a marked restoration of myelopoiesis. The proliferative advantage imposed on CD34+ cells by STAT5A1*6 depended on the down-modulation of C/EBPα, as reintroduction of C/EBPα induced a quick cell-cycle arrest and the onset of myeloid differentiation. Long-term culture–initiating cell (LTC-IC) frequencies were elevated from 0.8% ± 0.6% to 7.8% ± 1.9% by STAT5A1*6 as compared with controls, but these elevated LTC-IC frequencies were strongly reduced upon re-introduction of C/EBPα in STAT5A1*6 cells, and no second cobble-stone area–forming cells (CAFCs) could be generated from double-transduced cells. Enumeration of progenitors revealed that the number of colony-forming cells (CFCs) was reduced more than 20-fold when C/EBPα was co-expressed in STAT5A1*6 cells. Our data indicate that down-modulation of C/EBPα is a prerequisite for STAT5-induced effects on self-renewal and myelopoiesis.

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