STAT5 Is a Negative Regulator of Megakaryopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1182-1182
Author(s):  
Lyndsay Drayer ◽  
Sandra Olthof ◽  
Jan Jacob Schuringa ◽  
Edo Vellenga
Keyword(s):  
Critical Role ◽  
Negative Regulator ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Erythroid Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Cd34 Cells ◽  
Polyploid Cells

Abstract The transcription factor STAT5 plays a critical role in self-renewal and lineage commitment of hematopoietic stem cels (HSCs). We have recently shown in CB CD34+ cells that persistent activation of STAT5 results in enhanced self-renewal and induces erythroid differentiation, while myelopoiesis was severely impaired. In this study we analyzed the function of STAT5 during megakaryocyte (MK) differentiation. Using RNA interference we downregulated STAT5 expression in peripheral blood CD34+ cells. Cells were transduced with a lentiviral construct encoding eGFP and a short-hairpin RNA for STAT5 or with a control vector expressing YFP. Transduction efficiencies were determined by flow cytometric detection of eGFP/YFP and ranged from 40%–80%. Decreased STAT5 expression was verified by Western blot and quantitative RT-PCR (65% lower expression in STAT5 RNAi cells versus YFP+ control cells, p=<0.01). To assess the effects of decreased STAT5 expression on the progenitor pool, the transduced populations were sorted and enumerated in colony assays. Downregulation of STAT5 significantly increased the number of MK progenitors (1.9-fold, p=0.02) and resulted in a decrease of erythroid progenitors (0.6-fold, p<0.01), but did not affect the number of granulocyte/monocyte progenitors. Prospective isolation of immature and commited progenitors are currently being performed to distinguish if certain progenitor subsets are re-directed to the MK lineage by low STAT5 expression. Next, we analyzed the role of STAT5 during MK differentiation in unilineage suspension cultures. Transduced cells were cultured in serum-free medium containing TPO and SCF and scored for cell counts and expression of MK markers weekly. The percentage of transduced cells and their number did not differ when STAT5 RNAi cells were compared to controls, indicating that downregulation of STAT5 provides no proliferative advantage or disadvantage. However, the expression of glycoproteins IIb/IIIa (CD41) and Ib (CD42) was upregulated, and an increase in large, polyploid cells was observed in STAT5 RNAi cultures (15.2% polyploid cells versus 4.9% in control cells at day 7, p<0.01). RT-PCR analysis of transcription factors predominantly expressed in MK and erythroid lineages revealed that Runx1 and Erg were increased in STAT5 RNAi cells, and the level of Scl was decreased compared to control cells. These observations correlate with the increased MK differentiation observed in STAT5 downregulated cells. Together, these data demonstrate that the expression level of STAT5 has important effects regarding the type of hematopoietic cell development, with high levels favouring erythroid development and low levels enhancing MK differentiation and maturation.

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.

C1q Complement Cascade Protein as a Novel Modulator of the SDF-1-CXCR4 Axis and Hematopoietic Stem/Progenitor Cell Trafficking.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1212-1212
Author(s):  
Ali Jalili ◽  
Neeta Shirvaikar ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Peripheral Blood ◽  
Ex Vivo ◽  
Critical Role ◽  
Complement Cascade ◽  
Cell Trafficking ◽  
Immune Functions ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cxcr4 Axis

Abstract The first component of the complement (C) system C1q has multiple immune functions including acting as a chemoattractant for mature blood cells, and it has recently been reported that hematopoietic stem/progenitor cells (HSPC) express the C1q receptor, C1qRp. We have reported that the complement components C3 and C5 play an important role in the mobilization of HSPC and that G-CSF mobilization activates C by the classical Ig-dependent pathway (Blood2004;103:2071; Stem Cells 2007, in press). While C3 plays an important role in the retention of HSPC in BM, C5 is pivotal for their egress into peripheral blood. The aim of this study was to examine a possible role for C1q in the mobilization/homing of HSPC. We found (using RT-PCR and Western blotting) that the C1q gene and protein are strongly expressed in human mobilized peripheral blood (mPB) CD34+ cells, but not in steady-state bone marrow (BM) CD34+ cells. Importantly, we found that G-CSF stimulation of BM cells induces the expression of C1q in CD34+ cells, as detected by flow cytometry. Moreover, we demonstrated the presence of the receptor for C1q, C1qRp, in BM, mPB and cord blood (CB) CD34+ cells as well as in myeloid, megakaryocytic and erythroid progenitors ex vivo-expanded from CD34+ cells. The stromal derived factor (SDF)-1-CXCR4 axis plays a critical role in HSPC homing to BM and we showed using confocal microscopy that pre-incubation (for 1 h) of CB CD34+ cells on C1q-coated slides increased incorporation of the SDF-1 receptor, CXCR4, into membrane lipid rafts while C1q-coated filters increased chemoattraction (compared to bare filters) to an SDF-1 gradient. Although C1q itself is not a chemoattractant for HSPC, it primed the chemotactic response of CD34+ cells to a low SDF-1 gradient (10 ng/mL) to the extent that it approximated response to a high SDF-1 gradient (200 ng/mL), as well as the chemoinvasion of CB CD34+ cells across reconstituted basement membrane (Matrigel) to a low gradient of SDF-1, which was inhibited by the monoclonal anti-C1q antibody. In addition, C1q stimulated matrix-degrading metalloproteinase-9 secretion by CB CD34+ cells. In conclusion, we demonstrated for the first time that G-CSF mobilization strongly upregulates C1q in human HSPC which express the functional C1q receptor and that the first component of the complement cascade is involved in HSPC trafficking. This supports our previous data that C as an important modulator of the SDF-1-CXCR4 axis plays a pivotal role in the mobilization of HSPC.

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.

Decreased PU.1 and Enhanced CITED2 Cooperate To Maintain Self-Renewal In Hematopoietic Stem/Progenitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2411-2411
Author(s):  
Hein Schepers ◽  
Patrick M. Korthuis ◽  
Jan Jacob Schuringa ◽  
Edo Vellenga
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Fold Increase ◽  
Negative Regulator ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The transcriptional co-activator CITED2 has a conserved role in the maintenance of normal adult hematopoiesis. We have shown before that CD34+ cells from a subset of acute myeloid leukemia (AML) patients display enhanced CITED2 expression and that interfering with this expression is detrimental for leukemia maintenance. Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs) resulted in increased proliferation and skewed myelo-erythroid differentiation in vitro. Long-Term Culture-Initiating Cell assays (LTC-IC) revealed a 5-fold increase in the number of Cobblestone Area Forming Cells (CAFCs), as a result of an increase in the number of phenotypically defined CD34+CD38- HSCs. CFC frequencies were also enhanced 5-fold upon CITED2 overexpression. To further substantiate these observations in vivo, we transplanted CITED2-transduced CD34+ cells into NSG mice. CD34+ cells with increased CITED2 expression displayed a >10x higher engraftment at week 12, as compared to control cells, confirming the higher frequency of CD34+CD38- HSCs, while myelo-lymphoid differentiation of these cells was comparable to control transplanted cells. Till date we have not observed leukemia development in these transplanted mice, suggesting that CITED2 as a single hit is not sufficient to transform human CB CD34+ cells. We recently identified the myeloid transcription factor PU.1 as a strong negative regulator of CITED2 and enhanced CITED2 expression in AML samples correlates with low PU.1 expression. We therefore investigated whether high CITED2 and low PU.1 expression would collaborate in maintaining self-renewal of HSCs. We combined lentiviral downregulation of PU.1 with overexpression of CITED2 (PU.1Low-CITED2High) and performed LTC-IC cultures on MS5 stroma. These experiments revealed that combined loss of PU.1 and enhanced CITED2 expression was sufficient to induce a strong proliferative advantage compared to control cells. Furthermore, a 3-fold increase of progenitor numbers was observed in CFC assays. While overexpression of CITED2 alone was not sufficient to allow 2nd CFC formation, additional downregulation of PU.1 now led to colony formation upon serial replating. This replating capacity of PU.1Low-CITED2High cells was limited to CD34+CD38- HSCs, as replating of CD34+CD38+ progenitor cells did not yield CFCs. This suggests that the combined loss of PU.1 and enhanced CITED2 expression is sufficient to maintain self-renewal properties of HSC, but this combination is not sufficient to reinforce self-renewal in committed progenitor cells. To more stringently assess self-renewal, cells were first cultured for 4 weeks on MS5 under myeloid differentiating conditions (G-CSF, IL3 and TPO) and subsequently plated into CFC assays, followed by secondary and tertiary replating experiments. Only PU.1Low-CITED2High cells were able to form CFCs after 10 weeks of culture, indicating that this combination indeed preserves self-renewal. Current experiments focus on the in vivo engraftment and self-renewal properties of these PU.1Low-CITED2High cells. Preliminary data indicate that these PU.1Low-CITED2High cells contribute ∼3-fold more to the myeloid lineage at week 12, compared to control and CITED2 only cells, and AML development is currently being investigated in these mice. Together, these data suggest that CITED2 is sufficient to increase LTC-IC and CFC frequencies, to skew myeloid differentiation, and to enhance engraftment of CB CD34+ cells in xenograft mice. Furthermore, CITED2 overexpression together with reduced PU.1 levels is necessary to maintain stem cell self-renewal. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lower Hematopoietic Progenitor Cell Counts and Yields at Subsequent Donations Is Influenced By a Shorter Inter-Donation Interval between the First and Subsequent Mobilizations

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1962-1962
Author(s):  
Sandhya R. Panch ◽  
Brent R. Logan ◽  
Jennifer A. Sees ◽  
Bipin N. Savani ◽  
Nirali N. Shah ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Time Interval ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Peripheral Blood Cd34 ◽  
Cell Counts ◽  
Cd34 Cells

Introduction: Approximately 7% of unrelated hematopoietic stem cell (HSC) donors are asked to donate a subsequent time to the same or different recipient. In a recent large CIBMTR study of second time donors, Stroncek et al. incidentally found that second peripheral blood stem cell (PBSC) collections had lower total CD34+ cells, CD34+ cells per liter of whole blood processed, and CD34+ cells per kg donor weight. Based on smaller studies, the time between the two independent PBSC donations (inter-donation interval) as well as donor sex, race and baseline lymphocyte counts appear to influence CD34+ cell yields at subsequent donations. Our objective was to retrospectively evaluate factors contributory to CD34+ cell yields at subsequent PBSC donation amongst NMDP donors. Methods. The study population consisted of filgrastim (G-CSF) mobilized PBSC donors through the NMDP/CIBMTR between 2006 and 2017, with a subsequent donation of the same product. evaluated the impact of inter-donation interval, donor demographics (age, BMI, race, sex, G-CSF dose, year of procedure, need for central line) and changes in complete blood counts (CBC), on the CD34+ cell yields/liter (x106/L) of blood processed at second donation and pre-apheresis (Day 5) peripheral blood CD34+ cell counts/liter (x106/L) at second donation. Linear regression was used to model log cell yields as a function of donor and collection related variables, time between donations, and changes in baseline values from first to second donation. Stepwise model building, along with interactions among significant variables were assessed. The Pearson chi-square test or the Kruskal-Wallis test compared discrete variables or continuous variables, respectively. For multivariate analysis, a significance level of 0.01 was used due to the large number of variables considered. Results: Among 513 PBSC donors who subsequently donated a second PBSC product, clinically relevant decreases in values at the second donation were observed in pre-apheresis CD34+ cells (73.9 vs. 68.6; p=0.03), CD34+cells/L blood processed (32.2 vs. 30.1; p=0.06), and total final CD34+ cell count (x106) (608 vs. 556; p=0.02). Median time interval between first and second PBSC donations was 11.7 months (range: 0.3-128.1). Using the median pre-apheresis peripheral blood CD34+ cell counts from donation 1 as the cut-off for high versus low mobilizers, we found that individuals who were likely to be high or low mobilizers at first donation were also likely to be high or low mobilizers at second donation, respectively (Table 1). This was independent of the inter-donation interval. In multivariate analyses, those with an inter-donation interval of >12 months, demonstrated higher CD34+cells/L blood processed compared to donors donating within a year (mean ratio 1.15, p<0.0001). Change in donor BMI was also a predictor for PBSC yields. If donor BMI decreased at second donation, so did the CD34+cells/L blood processed (0.74, p <0.0001). An average G-CSF dose above 960mcg was also associated with an increase in CD34+cells/L blood processed compared to donors who received less than 960mcg (1.04, p=0.005). (Table 2A). Pre-apheresis peripheral blood CD34+ cells on Day 5 of second donation were also affected by the inter-donation interval, with higher cell counts associated with a longer time interval (>12 months) between donations (1.23, p<0.0001). Further, independent of the inter-donation interval, GCSF doses greater than 960mcg per day associated with higher pre-apheresis CD34+ cells at second donation (1.26, p<0.0001); as was a higher baseline WBC count (>6.9) (1.3, p<0.0001) (Table 2B). Conclusions: In this large retrospective study of second time unrelated PBSC donors, a longer inter-donation interval was confirmed to be associated with better PBSC mobilization and collection. Given hematopoietic stem cell cycling times of 9-12 months in humans, where possible, repeat donors may be chosen based on these intervals to optimize PBSC yields. Changes in BMI are also to be considered while recruiting repeat donors. Some of these parameters may be improved marginally by increasing G-CSF dose within permissible limits. In most instances, however, sub-optimal mobilizers at first donation appear to donate suboptimal numbers of HSC at their subsequent donation. Disclosures Pulsipher: CSL Behring: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding; Bellicum: Consultancy; Amgen: Other: Lecture; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medac: Honoraria. Shaw:Therakos: Other: Speaker Engagement.

scholarly journals Transplantation Induces Profound Changes in the Transcriptional Asset of Hematopoietic Stem Cells: Identification of Specific Signatures Using Machine Learning Techniques

2020 ◽  
Vol 9 (6) ◽  
pp. 1670
Author(s):  
Daniela Cilloni ◽  
Jessica Petiti ◽  
Valentina Campia ◽  
Marina Podestà ◽  
Margherita Squillario ◽  
...  
Keyword(s):  
Machine Learning ◽  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Machine Learning Techniques ◽  
Specific Gene ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

During the phase of proliferation needed for hematopoietic reconstitution following transplantation, hematopoietic stem/progenitor cells (HSPC) must express genes involved in stem cell self-renewal. We investigated the expression of genes relevant for self-renewal and expansion of HSPC (operationally defined as CD34+ cells) in steady state and after transplantation. Specifically, we evaluated the expression of ninety-one genes that were analyzed by real-time PCR in CD34+ cells isolated from (i) 12 samples from umbilical cord blood (UCB); (ii) 15 samples from bone marrow healthy donors; (iii) 13 samples from bone marrow after umbilical cord blood transplant (UCBT); and (iv) 29 samples from patients after transplantation with adult hematopoietic cells. The results show that transplanted CD34+ cells from adult cells acquire an asset very different from transplanted CD34+ cells from cord blood. Multivariate machine learning analysis (MMLA) showed that four specific gene signatures can be obtained by comparing the four types of CD34+ cells. In several, but not all cases, transplanted HSPC from UCB overexpress reprogramming genes. However, these remarkable changes do not alter the commitment to hematopoietic lineage. Overall, these results reveal undisclosed aspects of transplantation biology.

scholarly journals Successful hematopoietic stem cell mobilization and apheresis collection using plerixafor alone in sickle cell patients

2018 ◽  
Vol 2 (19) ◽  
pp. 2505-2512 ◽  
Author(s):  
Erica B. Esrick ◽  
John P. Manis ◽  
Heather Daley ◽  
Cristina Baricordi ◽  
Hélène Trébéden-Negre ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Standard Dose ◽  
Genetically Engineered ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Hematopoietic Stem Cell Mobilization ◽  
Cell Mobilization ◽  
Single Standard

Abstract Novel therapies for sickle cell disease (SCD) based on genetically engineered autologous hematopoietic stem and progenitor cells (HSPCs) are critically dependent on a safe and effective strategy for cell procurement. We sought to assess the safety and efficacy of plerixafor when used in transfused patients with SCD for HSC mobilization. Six adult patients with SCD were recruited to receive a single dose of plerixafor, tested at lower than standard (180 µg/kg) and standard (240 µg/kg) doses, followed by CD34+ cell monitoring in peripheral blood and apheresis collection. The procedures were safe and well-tolerated. Mobilization was successful, with higher peripheral CD34+ cell counts in the standard vs the low-dose group. Among our 6 donors, we improved apheresis cell collection results by using a deep collection interface and starting apheresis within 4 hours after plerixafor administration. In the subjects who received a single standard dose of plerixafor and followed the optimized collection protocol, yields of up to 24.5 × 106 CD34+ cells/kg were achieved. Interestingly, the collected CD34+ cells were enriched in immunophenotypically defined long-term HSCs and early progenitors. Thus, we demonstrate that plerixafor can be employed safely in patients with SCD to obtain sufficient HSCs for potential use in gene therapy.

Pum2 Protein Supports Maintenance and Suppresses Differentiation of Multipotent Hematopoietic Progenitors by Regulating the Function and Activation of c-kit Receptor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1692-1692
Author(s):  
Jie Yang ◽  
Danislav S. Spassov ◽  
Ronald G. Nachtman ◽  
Roland Jurecic
Keyword(s):  
Stem Cells ◽  
Rna Binding ◽  
Critical Role ◽  
Multilineage Differentiation ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Constitutive Activation ◽  
Gm Csf ◽  
Kit Receptor

Abstract Intrinsic mechanisms that regulate self-renewal of mammalian stem cells remain largely unknown. Stem cell maintenance and self-renewal in Drosophila and C. elegans are regulated by members of the conserved Pumilio family of RNA-binding proteins. We have previously described cloning and characterization of two mouse and human Pumilio genes (Pum1 and Pum2), which are abundantly transcribed in hematopoietic stem cells (HSC). To study the role of mammalian Pum proteins in HSC, Pum2 was over-expressed in a SCF-dependent multipotent progenitor cell line EML, which has the capacity for multilineage (erythroid, myeloid, B and T lymphoid) differentiation in vitro. In the presence of SCF EML cells undergo SCF-dependent self-renewal, thus remaining undifferentiated and retaining an immature phenotype. When cultured with hematopoietic cytokines (IL-3, GM-CSF, Epo, Tpo) EML cells differentiate into lineage-committed hematopoietic progenitors (e.g. granulocyte/macrophage (CFU-GM), burst-forming unit erythroid (BFU-E) and megakaryocytic (CFU-Meg) progenitors). Pum2 over-expression leads to uncoupling of the survival and differentiation signals in EML cells, and their SCF-independent maintenance. EML cells over-expressing Pum2 (Pum2-EML cells) also exhibit almost complete block of differentiation into multiple lineages in the absence of SCF. Moreover, although the culture with cytokine cocktail (IL-3, Epo, Tpo and GM-CSF) and retinoic acid enhances differentiation capacity of wild type EML cells, it was not sufficient to overcome the differentiation block in Pum2-EML cells. However, the repression of Pum2-EML cell differentiation is a reversible phenomenon, since the addition of SCF to Pum2-EML cell cultures, for at least 48 hours, restores their capacity to undergo multilineage differentiation and generate hematopoietic colonies. The SCF-independent maintenance of Pum2-EML cells seems to be caused by upregulated expression and constitutive activation of the SCF receptor c-kit, and is accompanied by constitutive activation of MAPK, PI3K and PLCγ signaling pathways in the absence of SCF. More importantly, Pum2-EML cells also exhibit upregulated expression and constitutive activation of a novel truncated form of c-kit receptor called tr-kit, which was found previously to be expressed preferentially in HSC. Tr-kit could play a critical role in the SCF-independent activation of the full-length c-kit receptor, leading to SCF-independent maintenance of Pum2-EML cells, and inhibition of their multilineage differentiation. The observation that Pum2-EML cells, maintained with or without SCF, are resistant to treatment with blocking anti-c-kit antibody (ACK2) and c-kit inhibitor STI-571, supports the notion that maintenance and survival of Pum2-EML cells in the presence of SCF is not due to an external activation of c-kit receptor through ligand binding. Taken together, these findings suggest a model in which survival and maintenance of multipotent hematopoietic progenitors are mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. In summary, mouse Pum2 protein could play an important role in supporting maintenance of HSC and multipotent progenitors through regulation of the SCF/c-kit signaling pathway, and could represent a part of the mechanism through which HSCs balance their self-renewal and commitment to differentiation.

Hematopoietic Origin of Fibroblasts and Their Precursors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 672-672 ◽  
Author(s):  
Yasuhiro Ebihara ◽  
Masahiro Masuya ◽  
Russell Owens ◽  
Su Yang ◽  
Richard P. Visconti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mononuclear Cells ◽  
Fluorescent Protein ◽  
Mesangial Cells ◽  
Pcr Analysis ◽  
Glomerular Mesangial Cells ◽  
General Belief ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Cd34 Cells

Abstract Recent studies indicate that hematopoietic stem cells (HSCs) are capable of reconstituting a number of non-hematopoietic organs and tissues. To define the potential of the HSC precisely, we initiated transplantation studies of a clonal population of cells derived from a single HSC. We used the bone marrow (BM) of transgenic enhanced green fluorescent protein (EGFP) mice as the source of donor HSCs. We demonstrated the HSC origin of glomerular mesangial cells (Masuya et al, Blood 101: 2215, 2003) and brain microglial cells and pericyte-like perivascular cells (Hess et al, Exp Neurol 186: 134, 2004). These observations and the fact that glomerular mesangial cells and pericytes are considered myofibroblasts suggested that fibroblasts are also derived from HSCs. In this abstract, we present evidence for HSC origin of fibroblasts and their precursors, BM fibroblast colony-forming units (CFU-F) and peripheral blood (PB) fibrocytes. Lin−Sca-1+c-Kit+CD34− cells from the BM of adult EGFP mice were individually sorted into 96 well Corning plates and cultured for 7 days in the presence of Steel factor and IL-11 or Steel factor and G-CSF. Viable clones consisting of fewer than 20 cells were individually transplanted into lethally irradiated mice. EGFP+ mononuclear cells were sorted from the BM cells of recipients showing high-level, multilineage hematopoietic reconstitution and assayed for CFU-F in Retronectin-treated tissue culture plates. Colonies consisting of more than 50 adherent cells were all EGFP+. The majority of the cells comprising the colonies were fibroblast-like, exhibited spindle-shaped or polygonal cytoplasm and had clear, ovoid nuclei. Flow cytometric analyses revealed that these cells expressed collagen-1 and discoidin domain receptor 2 (DDR2) and exhibited a decreased intensity of CD45. RT-PCR analysis of these cells revealed the presence of mRNA for procollagen 1 alpha-1, vimentin, fibronectin and DDR2. Next we analyzed the PB for donor origin fibrocytes, a fibroblast-like cell type that expresses both fibroblastic and hematopoietic phenotypes in culture. When nucleated PB cells from clonally engrafted mice were cultured on fibronectin-coated dishes, proliferation of EGFP+ fibroblast-like cells was detected. Only one-third of the EGFP+ cells expressed CD45 and most of the EGFP+ cells expressed both collagen-I and DDR2. Similar results were obtained with EGFP+ cells from mice transplanted with 100 uncultured Lin−Sca-1+c-Kit+CD34− cells or 1x106 BM nucleated cells. These studies excluded the effects of short-term culture on HSC differentiation and established the HSC origin of CFU-F and fibrocytes. Classic studies of CFU-F by Friedenstein and others have led to the general belief that mesenchymal stem cells (MSCs), rather than HSCs, generate a number of tissues including adipocytes, osteoblasts, chondrocytes, myocytes and vascular endothelial cells. Here we unequivocally demonstrated that CFU-F are of HSC origin, warranting a re-evaluation of the relationship between HSCs and MSCs.

Use of Chromatin Modifying Agents for Ex Vivo Expansion of Human Umbilical Cord Blood Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4208-4208
Author(s):  
Hiroto Araki ◽  
Nadim Mahmud ◽  
Mohammed Milhem ◽  
Mingjiang Xu ◽  
Ronald Hoffman
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Functional Properties ◽  
Ex Vivo ◽  
Fold Increase ◽  
Scid Mice ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The fixed number of hematopoietic stem cells (HSCs) within a single cord blood (CB) unit has limited the use of CB grafts for allogeneic transplantation in adults. Efforts to promote self-renewal and expansion of HSCs have been met with limited success. Using presently available ex-vivo culture techniques HSCs lose their functional properties in proportion to the number of cellular divisions they have undergone. We hypothesized that chromatin modifying agents, 5-aza-2′-deoxycytidine (5azaD) and histone deacetylase inhibitor, trichostatin A (TSA) could reactivate pivotal genes required for retaining the functional properties of dividing HSC. We have demonstrated previously that the fate of human bone marrow CD34+ cells could be altered by the addition of 5azaD/TSA (Milhem et al. Blood.2004;103:4102). In our current studies we hypothesized that in vitro exposure of CB CD34+ cells to chromatin modifying agents might lead to optimal HSC expansion to permit transplantation of adults. A 12.5-fold expansion was observed in the 5azaD/TSA treated CD34+CD90+ cell cultures containing SCF, thrombopoietin and FLT3 ligand (cytokines) in comparison to the input cell number. Despite 9 days of culture, 35.4% ± 5.8% (n = 10) of the total cells in the cultures exposed to chromatin modifying agents were CD34+CD90+ as compared to 1.40 % ± 0.32% in the culture containing cytokines alone. The 12.5-fold expansion of CD34+CD90+ cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold expansion of cobblestone area-forming cells (CAFC). The frequency of SCID repopulating cells (SRC) was 1 in 26,537 in primary CB CD34+CD90+ cells but was increased to 1 in 2,745 CD34+CD90+ cells following 9 days of culture in the presence of 5azaD/TSA resulting in a 9.6-fold expansion of the absolute number of SRC. In contrast, the cultures lacking 5azaD/TSA had a net loss of both CFC/CAFC as well as SRC. The expansion of cells maintaining CD34+CD90+ phenotype was not due to the retention of a quiescent population of cells since all of the CD34+CD90+ cells in the culture had undergone cellular division as demonstrated by labeling with a cytoplasmic dye. CD34+CD90+ cells that had undergone 5–10 cellular divisions in the presence of 5azaD/TSA but not in the absence still retained the ability to repopulate NOD/SCID mice. 5azaD/TSA treated CD34+CD90+ cells, but not CD34+CD90- cells were responsible for in vivo hematopoietic repopulation of NOD/SCID assay, suggesting a strong association between CD34+CD90+ phenotype and their ability to repopulate NOD/SCID mice. We next assessed the effect of 5azaD/TSA treatment on the expression of HOXB4, a transcription factor which has been implicated in HSC self-renewal. A significantly higher level of HOXB4 protein was detected by western blot analysis after 9 days of culture in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. The almost 10-fold increase in SRC achieved using the chromatin modifying agents should be sufficient to increase the numbers of engraftable HSC within a single human CB unit so as to permit these expanded grafts to be routinely used for transplanting adult recipients.

Sign in / Sign up

Export Citation Format

Share Document