Microrna Mediated Regulation of Hematopoietic Stem Cell Aging

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 602-602 ◽  
Author(s):  
Safak Yalcin ◽  
Mark Carty ◽  
Joseph Yusup Shin ◽  
Richard A Miller ◽  
Christina Leslie ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Fold Increase ◽  
Erythroid Progenitors ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Age Related ◽  
Old Mice

Abstract Aging hematopoietic stem cells (HSCs) exhibit numerous functional alterations including reduced capacity for self-renewal, myeloid-biased differentiation, and reduced production of mature lymphocytes and red blood cells. Interventions such as calorie restriction (CR) and rapamycin (Rapa) treatment have been shown to increase lifespan and to delay the onset of age-related diseases, and some studies have demonstrated that they may improve HSC function through poorly defined mechanisms. We and others have shown that microRNAs (miRNAs) are potent cell-intrinsic regulators of HSC self-renewal and lineage specification and also contribute to age-related disorders such as acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS). We hypothesized that miRNAs may underlie the recovery of HSC function observed in anti-aging mouse models, and thus we characterized miRNA expression profiles from HSCs (Lin-c-Kit+Sca-1+CD34-CD150+) from young mice (12-16 weeks old), old mice (20-22 months), and old mice that had been treated with anti-aging interventions. Evaluation of HSCs from CR and Rapa treated old mice revealed numerous changes consistent with inhibition/reversal of age-related HSC changes including a 5-fold reduction in HSC frequency (p=0.04), 2-fold increase in erythroid progenitors (pro-erythroblasts, p=0.04), 2.5 fold increase in common lymphoid progenitors (CLP; Lin-c-Kit+Sca-1+CD127+FLK2+, p=0.05), as well as 3.5-fold increase in peripheral blood B cells (p=0.002), 2.2 fold decrease in platelets (p=0.01), and increased red blood cells (p=0.04). These changes were associated with statistically significant increases in the percentage of HSCs in S/M/G2 (p=0.045), and undergoing apoptosis (p=0.05). Using a TaqMan-based qPCR expression profiling method evaluating 750 miRNAs, we found that old HSCs exhibited altered expression of 91 miRNAs compared to young (FDR <0.1, P <0.05). Moreover, HSCs from both CR and Rapa treated old mice exhibited expression of 60 miRNAs at levels similar to young, normal HSCs. miR-125b, a miRNA we and others previously showed to positively regulate HSC self-renewal, was reduced 2.2-fold in old mice, and its expression was restored in CR and Rapa treated HSCs. Lentivirally mediated expression of miR-125b in old HSCs increased their long-term reconstitution capacity 8.1-fold compared to control old HSCs based on donor chimerism levels at 16 weeks post-transplantation, resulting in chimerism levels similar to mice transplanted with young HSCs expressing miR-125b. The improved HSC engraftment capacity of old HSCs transduced with miR-125b was accompanied by statistically significant increases in the frequencies of lymphoid biased HSCs (Lin-c-Kit+Sca-1+CD34-CD150neg-low), megakaryocyte-erythroid progenitors (MEPs), CLPs, and peripheral blood B- and T-cells, compared to old HSCs transduced with control lentivirus (p<0.05 for all indicated cell types). While enforced expression of high levels of miR-125b in mouse HSPCs has been reported to induce myeloid leukemias, there was no evidence of a hematologic malignancy in mice transplanted with miR-125b transduced old HSCs up to 6 months post-transplantation. Overall, these results demonstrate that functional HSC aging phenotypes can be that inhibited/reversed by anti-aging interventions, that miR-125b regulates HSC aging, and that anti-aging interventions may exert their positive effects on HSC function by regulating miR-125b expression. Disclosures No relevant conflicts of interest to declare.

Human CD34+Cells Mobilized by AMD3100 Demonstrate Enhanced NOD/SCID Repopulating Function Compared to CD34+ Cells Mobilized by Granulocyte Colony Stimulating Factor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1962-1962 ◽  
Author(s):  
David A. Hess ◽  
Louisa Wirthlin ◽  
Timothy P. Craft ◽  
Jesper Bonde ◽  
Ryan W. Lahey ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Paired Comparison ◽  
Fold Increase ◽  
Human Cells ◽  
Lymphoid Cells ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Interactions between stromal derived factor-1 (SDF-1 or CXCL12), and its receptor CXCR4 regulate hematopoietic stem and progenitor cell retention in the bone marrow. AMD3100, a bicyclam molecule that selectively blocks the interaction between CXCL12 and CXCR4, has recently been used in clinical trials to rapidly mobilize hematopoietic progenitor cells. However, the functional properties of human stem and progenitor cells mobilized with this agent are not well characterized. Here, we directly compared the NOD/SCID repopulating function of CD34+ cells rapidly mobilized (4 hours) by AMD3100 versus CD34+ cells mobilized after 5 days of G-CSF treatment. A total of 7 HLA-matched sibling donors were leukapheresed after a single injection of 240ug/kg AMD3100. After 1 week of drug clearance, the same donor was mobilized with G-CSF, allowing a paired comparison of the repopulating function of cells mobilized by the two agents. Total CD34+ cells mobilized by AMD3100 treatment averaged 1.2±0.4x106 CD34+ cells/kg (range 0.4–2.1x106 CD34+ cells/kg), as compared to G-CSF treatment at 3.2±0.9x106 CD34+ cells/kg (range 1.7–5.7 x106 CD34+ cells/kg). Leukapheresis total mononuclear cell (MNC) fraction or purified CD34+ cells (>90% purity), were isolated and transplanted into sublethally irradiated NOD/SCID mice at varying doses. BM, spleen, and peripheral blood of mice were harvested 7–8 weeks post-transplantation and analyzed by flow cytometry for the presence or absence of engrafting human cells. Low frequency human engraftment events (<0.2% human cells) were confirmed by PCR for P17H8 alpha-satellite human DNA sequences. Injection of 1–40x106 MNC or 0.5–5x105 CD34+ cells produced consistent human engraftment and allowed limiting dilution analysis using Poisson statistics to be performed on paired samples of AMD3100 and G-CSF leukapheresis products from 3 individual patients. The calculated frequencies of NOD/SCID repopulating cells (SRC) were 1 SRC in 11.5x106 AMD3100-mobilized MNC (n=50) compared to 1 SRC in 44.8x106 G-CSF-mobilized MNC (n=55). For purified CD34+ populations, the overall frequency of repopulating cells was 1 SRC in 1.0x105 AMD3100-mobilized CDC34+ cells (n=53) compared to 1 SRC in 3.1x105 G-CSF-mobilized CD34+ cells (n=45). These data correspond to a 3–4-fold increase in overall repopulating function demonstrated by AMD3100 mobilized cells. Multilineage hematopoietic differentiation of transplanted CD34+ cells was similar for AMD3100 and G-CSF-mobilized CD34+ cells, with equivalent production of myelo-monocytic cells (CD33+CD14+), immature B-lymphoid cells (CD19+CD20+), and primitive repopulating (CD34+CD133+CD38−) cells 7–8 weeks post-transplantation. These studies indicate that human AMD3100-mobilized MNC and purified CD34+ cells possess enhanced repopulating capacity, as compared to G-CSF mobilized counterparts from the same donor. Thus, AMD3100 mobilized peripheral blood represents a rapidly obtained and highly functional source of repopulating hematopoietic stem cells for clinical transplantation procedures.

scholarly journals Disruption of the Normal Hematopoietic Hierarchy Leading to Stem Cell Exhaustion in an Animal Model of Myelodysplastic Syndrome

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1698-1698
Author(s):  
Yang Jo Chung ◽  
Peter D. Aplan
Keyword(s):  
Stem Cell ◽  
Myelodysplastic Syndrome ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Brdu Incorporation ◽  
Compensatory Mechanism ◽  
Self Renewal ◽  
Hematopoietic Stem

The ineffective hematopoiesis that is characteristic of myelodysplastic syndrome (MDS) suggests functional defects of hematopoietic stem and progenitor cells (HSPC). NUP98-HOXD13 (NHD13) transgenic mice recapitulate many features of human MDS such as ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and transformation to acute myeloid leukemia (AML), and have been used as a pre-clinical model for human MDS. NHD13 mice universally develop signs of MDS (e.g., peripheral blood cytopenia, macrocytosis, dysplasia) at approximately 5 months of age, with median survival of 10 months. Two month old NHD13 mice do not show clear evidence of MDS such as peripheral blood cytopenia, dysplasia, or transformation to AML. Bone marrow nucleated cells (BMNC) from two month old NHD13 mice have a modest 1.3-fold increase of lineage negative (LN) BMNCs compared to age matched WT mice. The increased number of LN BMNCs appeared to be primarily due to a 3.4-fold increase of the LN Sca-1+cKit-(LS+Kˉ) cells, an early lymphoid-committed precursor. Lineage negative Sca-1+ c-Kit+ (LSK) cells, which include the most immature, undifferentiated cells, can be divided into five sub populations, based on expression of Flk2, CD150, and CD48. These populations have been designated Long-Term Hematopoietic Stem Cell (LT-HSC), Short-Term HSC, (ST-HSC), and Multi-Potent Progenitor 2, 3, and 4 (MPP2, MPP3, and MPP4) based on functional assays. Two-month old NHD13 mice had decreased MPP4 (5-fold), decreased LT-HSC (3.6-fold) and increased ST-HSC (2.3-fold) compared with the age matched WT mice. The expansion of ST-HSC two-month old NHD13 mice was associated with increased cell proliferation of ST- HSC, as assessed by bromo-deoxy-uridine (BRDU) incorporation. We next studied LSK subsets from NHD13 mice aged seven months, which coincided with peripheral blood findings consistent with MDS (e.g. anemia, thrombocytopenia, macrocytosis), BM from seven month old NHD13 mice showed significant reductions of all LSK population subsets. LT-HSCs show differential expression of the CD41 antigen, and CD41ˉ LT-HSCs are more quiescent than CD41+ LT-HSCs and are thought to reside at the apex of the hematopoietic differentiation hierarchy. Although there was no difference in the absolute number of quiescent CD41ˉ LT-HSC between two and six month old WT mice, six month old NHD13 mice show a marked decrease (4.2 fold) in CD41ˉ LT-HSCs, suggesting exhaustion of LT-HSC in NHD13 mice. Colony forming assays were used to assess function of the five LSK sub-populations in vitro. LT-HSC and ST- HSC from NHD13 BMNC did not produce any colonies in two independent experiments, whereas MPP2 and MPP3 from NHD13 BMNC produced a similar number and lineage distribution of colonies compared to WT BMNC. This result suggested that HSCs from NHD13 BMNC may be functionally impaired, and that NHD13 hematopoietic progenitor cells may instead be derived primarily from MPP2 and MPP3. To evaluate HSC self-renewal activity, the five LSK subsets from NHD13 BMNC were transplanted to lethally irradiated mice together with 5 x 105 WT BMNC competitor cells. None of the NHD13 LSK sub-populations showed evidence of engraftment. Since NHD13 LN BMNC have previously been shown to be more prone to apoptosis than their WT counterpart, it is possible that lack of engraftment of NHD13 LSK subsets was due to the ex vivo sorting procedure. However, we also considered the possibility that NHD13 lineage positive (LP) BMNC had acquired self-renewal potential, and were contributing to long term hematopoiesis in the NHD13 BM. Therefore, we transplanted LP and LN BMNC from NHD13 or WT mice into WT recipients, again with WT competitor BMNC. Almost half of the NHD13 LP recipients showed long-term (>26 weeks) myeloid engraftment, whereas none of the WT LP recipients showed long term myeloid engraftment. Taken together, these findings suggest that the primitive LT-HSC (LSK Flk2ˉ CD150+CD48ˉ CD41ˉ) from NHD13 BM become exhausted with age, corresponding to the presentation of findings consistent with MDS (peripheral blood cytopenia, macrocytosis). Furthermore, self-renewal activity of NHD13 LP BMNCs suggest the existence of a compensatory mechanism for the homeostasis of hematopoiesis in MDS. Disclosures Aplan: NIH: Patents & Royalties: royalties for the invention of NUP98-HOXD13.

Erythroid Lineage-Restricted Expression of Jak2V617F Is Sufficient to Induce a Myeloproliferative Disease in Mice,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3861-3861
Author(s):  
Hajime Akada ◽  
Saeko Hamada ◽  
Golam Mohi
Keyword(s):  
Stem Cells ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Myeloproliferative Neoplasms ◽  
Jak2v617f Mutation ◽  
Target Cells ◽  
Dependent Manner ◽  
Myeloproliferative Disease ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem

Abstract Abstract 3861 A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in most cases of Ph-negative myeloproliferative neoplasms (MPNs) including ∼95% patients with polycythemia vera (PV) and 50–60% patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). To investigate the contribution of JAK2V617F in MPNs, we generated a conditional Jak2V617F knock-in mouse (Akada et al., Blood 2010; 115: 3589–3597). Expression of Jak2V617F in all hematopoietic compartments including the hematopoietic stem cells (HSC) resulted in a PV-like disease associated with a marked expansion of erythroid progenitors in the bone marrow and spleen. Since Jak2 is essential for normal erythropoiesis and expression of Jak2V617F mutant enhances erythropoiesis, so we asked if erythroid progenitors are actual target cells for Jak2V617F mutation. To address this question, we have specifically expressed Jak2V617F in erythroid progenitors using the EpoR-Cre mice. Expression of heterozygous Jak2V617F in erythroid progenitors resulted in a polycythemia-like phenotype characterized by increase in hematocrit and hemoglobin, increased red blood cells, Epo-independent erythroid colonies, and splenomegaly. Erythroid lineage-specific expression of homozygous Jak2V617F resulted in significantly greater increase in hematocrit, hemoglobin, red blood cells, Epo-independent erythroid colonies, and splenomegaly compared to heterozygous Jak2V617F expression. These results suggest that erythroid lineage-restricted expression of Jak2V617F is sufficient to induce a polycythemia-like disease in a gene-dose dependent manner. However, transplantation of Jak2V617F-expressing erythroid progenitors (c-kithighTer119lowCD71high or c-kitlowTer119highCD71high) from the diseased mice into lethally irradiated recipients could not transfer the disease suggesting that Jak2V617F mutation does not confer self-renewal capacity to erythroid progenitors. We also observed that only Jak2V617F-expressing HSC has the unique capacity to serially transplant the myeloproliferative disease in mice. Taken together, our results suggest that HSCs are the disease-initiating cancer stem cells and erythroid progenitors are the target cells in Jak2V617F-evoked MPN. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Glutamylation of deubiquitinase BAP1 controls self-renewal of hematopoietic stem cells and hematopoiesis

2019 ◽  
Vol 217 (2) ◽  
Author(s):  
Zhen Xiong ◽  
Pengyan Xia ◽  
Xiaoxiao Zhu ◽  
Jingjing Geng ◽  
Shuo Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Peripheral Blood Cells ◽  
Self Renewal ◽  
Hematopoietic Stem

All hematopoietic lineages are derived from a limited pool of hematopoietic stem cells (HSCs). Although the mechanisms underlying HSC self-renewal have been extensively studied, little is known about the role of protein glutamylation and deglutamylation in hematopoiesis. Here, we show that carboxypeptidase CCP3 is most highly expressed in BM cells among CCP members. CCP3 deficiency impairs HSC self-renewal and hematopoiesis. Deubiquitinase BAP1 is a substrate for CCP3 in HSCs. BAP1 is glutamylated at Glu651 by TTLL5 and TTLL7, and BAP1-E651A mutation abrogates BAP1 glutamylation. BAP1 glutamylation accelerates its ubiquitination to trigger its degradation. CCP3 can remove glutamylation of BAP1 to promote its stability, which enhances Hoxa1 expression, leading to HSC self-renewal. Bap1E651A mice produce higher numbers of LT-HSCs and peripheral blood cells. Moreover, TTLL5 and TTLL7 deficiencies sustain BAP1 stability to promote HSC self-renewal and hematopoiesis. Therefore, glutamylation and deglutamylation of BAP1 modulate HSC self-renewal and hematopoiesis.

Expression of a HoxC4 Retroviral Vector Mediates Expansion of Murine Hematopoietic Stem Cells with Normal Hematopoiesis in Transplanted Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2788-2788
Author(s):  
Lilia Stepanova ◽  
Brian P. Sorrentino
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow

Abstract Homeobox (Hox) transcription factors are important regulators of hematopoietic cell proliferation and differentiation. Of them, HoxB4 is of particular interest because overexpression promotes rapid expansion of mouse hematopoietic stem cells (HSCs) without causing neoplastic transformation. Despite the effects of HoxB4 overexpression on HSCs, mice that are homozygous for HoxB4 gene deletion have only subtle defects in HSCs and progenitor cells. We hypothesized that other paralogs of HoxB4 may also be capable of inducing HSC expansion could thereby compensate for loss of HoxB4 function. To test this hypothesis, we have studied the effects of retroviral overexpression of a HoxC4 gene in murine progenitors and HSCs. The murine HoxC4 cDNA was cloned and inserted into an MSCV vector that co-expresses an IRES-YFP reporter gene. We transduced murine bone marrow cells with a MSCV-HoxC4-YFP vector and compared the secondary replating efficiency of myeloid colonies (CFU-Cs) to that seen using either a MSCV-HoxB4-GFP or an MSCV-GFP vector. This assay tests for progenitor cell self-renewal which is increased using HoxB4-expressing vectors. Cells transduced with the MSCV-HoxC4-YFP vector formed 20–40 times more secondary CFU-Cs than with cells transduced with the MSCV-GFP control vector. This increase in CFU-C replating efficiency was equivalent to that seen with the MSCV-HoxB4-IRES-GFP vector. To test the in vivo effects of the MSCV-HoxC4-YFP vector on self-renewal of HSCs, we transplanted lethally irradiated mice with a mixture of cells; 20% transduced with the MSCV-HoxC4-YFP vector and 80 % mock-transduced. Peripheral blood analysis of the transplanted recipients up to 28 weeks post-transplantation showed that the percentage of cells transduced with the MSCV-HoxC4-YFP vector was 70–85% in both lymphoid and myeloid cells in the peripheral blood. A similar degree of chimerism was noted in concurrent controls using the MSCV-HoxB4-GFP vector. In contrast, the percentages of peripheral blood cells transduced with the MSCV-GFP vector was only 15–25%, paralleling the input ratios of transplanted cells. Secondary transplantation experiments showed stable levels of chimerism in both HoxC4 and HoxB4 groups, indicating that the expansion seen with the MSCV-HoxC4-YFP vector occurred at the HSC level. These results indicate that retroviral-mediated expression of HoxC4, like HoxB4, can cause significant expansion of HSCs in vivo. Because several other Hox genes can cause hematopoietic abnormalities and leukemia when expressed from a retroviral vector, we transplanted lethally irradiated mice with 4x106 cells that were transduced with the MSCV-HoxC4-YFP vector and monitored the animals for survival and complete blood counts. Now, at 33 weeks post transplantation, no tumor formation was observed in mice expressing either the HoxB4 or the HoxC4 vector, and peripheral blood counts have remained normal. Our results show that retroviral overexpression of HoxC4 can induce a significant expansion of the HSCs in vivo, and suggest that expression of HoxC4 may compensate for the loss of HoxB4 in knockout mice. We are currently analyzing the effects of HoxA4 and HoxD4 to determine if they share the same functional characteristics, and are also determining whether HoxB4 and HoxC4 are modulating the same downstream genes using microarray analysis of transduced murine bone marrow cells.

Pleiotrophin Is a Growth Factor for Hematopoietic Stem Cells and Induces Stem Cell Self-Renewal

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 78-78
Author(s):  
Heather A Himburg ◽  
Garrett Muramoto ◽  
Sarah Kristen Meadows ◽  
Alice Bryn Salter ◽  
Nelson J Chao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Hematopoietic Stem Cells ◽  
Fold Increase ◽  
Post Transplantation ◽  
Cell Content ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
A Genome ◽  
Cells Cultured

Abstract The ability to undergo self-renewal is a defining feature of hematopoietic stem cells (HSCs) but the extrinsic signals which regulate HSC self-renewal remain unclear. We performed a genome-wide expression analysis on primary human brain ECs (HUBECs, n=10) which support the ex vivo expansion of HSCs in non-contact culture (Blood100:4433–4439; Blood105:576–583) and non-brain ECs which do not support HSC expansion (n=8) in order to identify soluble proteins overexpressed by the HSC-supportive HUBECs. We identified pleiotrophin (PTN), an 18 kD heparin binding growth factor, to be 32-fold overexpressed in HUBECs as compared to non-supportive EC lines. PTN has established activity in angiogenesis, embryogenesis, neuronal cell growth and tumorigenesis, but has no known function in hematopoiesis. We first tested whether secreted PTN was responsible for the amplification of HSCs that we have observed in co-cultures of HSCs with HUBECs via “loss of function” studies in which a blocking anti-PTN antibody was added to HUBEC cultures and HSC content was measured. Competitive repopulating unit (CRU) assays were performed in which limiting doses of donor CD45.1+ bone marrow (BM) 34−c-kit+sca-1+lin− (34-KSL) HSCs (10, 30 or 100 cells) or their progeny following 7 day non-contact culture with HUBECs + IgG or HUBECs + a blocking anti-PTN were transplanted into lethally irradiated CD45.2+ C57Bl6 mice. Mice transplanted with the progeny of 34-KSL cells cultured with HUBECs demonstrated 4–6 fold increased levels of donor-derived CD45.1+ multilineage repopulation at 8-, 12- and 24-weeks post-transplantation as compared to mice transplanted with input 34-KSL cells. In contrast, mice transplanted with the progeny of 34-KSL cells following culture with HUBECs + anti-PTN demonstrated significant reduction in donor CD45.1+ cell repopulation compared to mice transplanted with the progeny of HUBEC cultures and no difference in donor CD45.1+ cell engraftment compared to mice transplanted with input 34-KSL cells. CRU frequency within day 0 34-KSL cells was estimated to be 1 in 40 cells (95% Confidence Interval [CI]: 1/22-1/72), whereas the CRU estimate within the progeny of 34-KSL cells following HUBEC culture was 1 in 4 cells (CI: 1/2-1/9). The addition of anti-PTN to the HUBEC co-culture decreased the CRU estimate to 1 in 29 cells (CI: 1/16-1/52), suggesting that PTN signaling was responsible for the expansion of HSCs observed in HUBEC co-cultures. In order to confirm whether PTN is indeed a novel growth and self-renewal factor for HSCs, we next performed “gain of function” studies in which 34-KSL cells were placed in liquid suspension cultures with cytokines (thrombopoietin 50 ng/mL, SCF 120 ng/mL, flt-3 ligand 20 ng/mL) with and without the addition of increasing doses of recombinant murine PTN (10, 50 and 100 ng/mL) and total cell expansion and HSC content were compared. The addition of 100 ng/mL PTN to cytokine cultures caused a 20-fold increase in KSL cell content at day 7 compared to input (P<0.001), whereas a decline in KSL cells was observed with cytokine cultures alone (P<0.001), suggesting that PTN caused an expansion of stem/progenitor cells in vitro. Competitive repopulating assays were performed in which CD45.2+ recipient mice were lethally irradiated and transplanted with limiting doses (10, 30 and 100 cells) of CD45.1+ donor BM 34-KSL cells or their progeny following culture with cytokines alone or cytokines + 100 ng/mL PTN. CRU analysis at 4 weeks post-transplantation revealed that the CRU frequency within input 34-KSL cells was was 1 in 32 cells (CI: 1/18-1/57) and the CRU estimate within the progeny of 34-KSL cells cultured with cytokines alone was 1 in 69 (CI: 1/36-1/130). Conversely, the CRU estimate within the progeny of 34-KSL cells cultured with cytokines + PTN was 1 in 4 cells (CI: 1/2-1/10), indicating a 8-fold increase in short term repopulating cell content in response to PTN treatment. Longer term analysis will be performed in these mice to confirm whether PTN treatment induces the self-renewal and amplification of long-term repopulating HSCs in culture. Taken together, these data demonstrate that secreted PTN is primarily responsible for amplification of HSCs that we have observed in cultures of HSCs with ECs and the addition of PTN alone induces the expansion of phenotypic and functional HSCs in culture. PTN is therefore a novel soluble growth factor for HSCs and appears to play an important role in the extrinsic regulation of HSC self-renewal.

Differential Expression of c-Kit Identifies Hematopoietic Stem Cells with Variable Self-Renewal Potential.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2325-2325
Author(s):  
Joseph Yusup Shin ◽  
Wenhuo Hu ◽  
Christopher Y. Park
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Differential Expression ◽  
Peripheral Blood ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 2325 Hematopoietic stem cells (HSC) can be identified on the basis of differential cell surface protein expression, such that 10 out of 13 purified HSC (Lin−c-Kit+Sca-1+CD150+CD34−FLK2−) exhibit long-term reconstitution potential in single-cell transplants. HSCs express c-Kit, and interactions between c-Kit and its ligand, stem cell factor, have been shown to be critical for HSC self-renewal; however, HSCs express a log-fold variation in c-Kit levels. We hypothesized that differing levels of c-Kit expression on HSCs may identify functionally distinct classes of HSCs. Thus, we measured the function and cellular characteristics of c-Kithi HSCs and c-Kitlo HSCs (defined as the top 30% and bottom 30% of c-Kit expressors, respectively), including colony formation, cell cycle status, lineage fates, and serial engraftment potential. In methylcellulose colony assays, c-Kithi HSCs formed 5-fold more colonies than c-Kitlo HSCs (P=0.01), as well as 4-fold more megakaryocyte colonies in vitro. c-Kithi HSC were 2.4-fold enriched for cycling cells (G2-S-M) in comparison to c-Kitlo HSC as assessed by flow cytometry in vivo (15.4% versus 6.4%, P=0.001). Lethally irradiated mice competitively transplanted with 400 c-Kitlo HSCs and 300,000 competitor bone marrow cells exhibited increasing levels of donor chimerism, peaking at a mean of 80% peripheral blood CD45 chimerism by 16 weeks post-transplantation, whereas mice transplanted with c-Kithi HSCs reached a mean of 20% chimerism (p<0.00015). Evaluation of the bone marrow revealed an increase in HSC chimerism from 23% to 44% in mice injected with c-Kitlo HSCs from weeks 7 to 18, while HSC chimerism decreased from 18% to 3.0% in c-Kithi HSC-transplanted mice (P<0.00021). Levels of myeloid chimerism in the bone marrow and peripheral blood were not significantly different during the first 4 weeks following transplantation between mice transplanted with c-Kithi or c-Kitlo HSCs, and evaluation of HSC bone marrow lodging at 24 hours post-transplantation demonstrated no difference in the number of c-Kithi and c-Kitlo HSCs, indicating that differential homing is not the reason for the observed differences in long-term engraftment. Donor HSCs purified from mice transplanted with c-Kithi HSC maintained higher levels of c-Kit expression compared to those from mice injected with c-Kitlo HSC by week 18 post-transplantation (P=0.01). Secondary recipients serially transplanted with c-Kithi HSC exhibited a chimerism level of 40% to 3% from week 4 to 8 post-secondary transplant, whereas chimerism levels remained at 6% in mice injected with c-Kitlo HSC. These results indicate that c-Kithi HSCs exhibit reduced self-renewal capacity compared with c-Kitlo HSCs, and that the differences in c-Kithi and c-Kitlo HSC function are cell-intrinsic. Analysis of transplanted HSC fates revealed that c-Kithi HSCs produced two-fold more pre-megakaryocyte-erythroid progenitors and pluriploid megakaryocytes compared to their c-Kitlo counterparts in vivo, suggesting a megakaryocytic lineage bias in c-Kithi HSC. Consistent with this finding, the transplanted c-Kithi HSC gave rise to 10-fold more platelets and reached a maximum platelet output two days earlier than c-Kitlo HSC. To determine the potential mechanisms underlying the transition from c-Kitlo to c-Kithi HSCs, we assessed the activity of c-Cbl, an E3 ubiquitin ligase known to negatively regulate surface c-Kit expression in a Src-dependent manner. Flow cytometric analysis revealed 6-fold more activated c-Cbl in freshly purified c-Kitlo HSC compared to c-Kithi HSC (P=0.02), suggesting that functional loss of c-Cbl increases c-Kit expression on c-Kitlo HSCs. Mice treated for nine days with Src inhibitors, which inhibit c-Cbl activity, experienced a 1.5-fold and 2-fold increase in the absolute number of c-Kithi HSCs (P=0.067) and megakaryocyte progenitors (P=0.002), respectively. Thus, c-Cbl loss likely promotes the generation of c-Kithi HSCs. In summary, differential expression of c-Kit identifies HSC with distinct functional attributes with c-Kithi HSC exhibiting increased cell cycling, megakaryocyte lineage bias, decreased self-renewal capacity, and decreased c-Cbl activity. Since c-Kitlo HSC represent a population of cells enriched for long-term self-renewal capacity, characterization of this cell population provides an opportunity to better understand the mechanisms that regulate HSC function. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Functional but Abnormal Adult Erythropoiesis in the Absence of the Stem Cell Leukemia Gene

2005 ◽  
Vol 25 (15) ◽  
pp. 6355-6362 ◽  
Author(s):  
Mark A. Hall ◽  
Nicholas J. Slater ◽  
C. Glenn Begley ◽  
Jessica M. Salmon ◽  
Leonie J. Van Stekelenburg ◽  
...  
Keyword(s):  
Stem Cell ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Target Genes ◽  
Growth Defect ◽  
Unexpected Finding ◽  
Cell Leukemia ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Stem Cell Leukemia

ABSTRACT Previous studies have indicated that the stem cell leukemia gene (SCL) is essential for both embryonic and adult erythropoiesis. We have examined erythropoiesis in conditional SCL knockout mice for at least 6 months after loss of SCL function and report that SCL was important but not essential for the generation of mature red blood cells. Although SCL-deleted mice were mildly anemic with increased splenic erythropoiesis, they responded appropriately to endogenous erythropoietin and hemolytic stress, a measure of late erythroid progenitors. However, SCL was more important for the proliferation of early erythroid progenitors because the predominant defects in SCL-deleted erythropoiesis were loss of in vitro growth of the burst-forming erythroid unit and an in vivo growth defect revealed by transplant assays. With respect to erythroid maturation, SCL-deleted proerythroblasts could generate more mature erythroblasts and circulating red blood cells. However, SCL was required for normal expression of TER119, one of the few proposed target genes of SCL. The unexpected finding that SCL-independent erythropoiesis can proceed in the adult suggests that alternate factors can replace the essential functions of SCL and raises the possibility that similar mechanisms also explain the relatively minor defects previously observed in SCL-null hematopoietic stem cells.

Self-Renewal of Hematopoietic Stem Cells by a Small Molecule

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 210-210
Author(s):  
Anthony E Boitano ◽  
Peter G Schultz ◽  
Michael Cooke
Keyword(s):  
Cord Blood ◽  
Small Molecule ◽  
High Throughput ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Fold Increase ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Hematopoietic stem cell (HSC) transplantation has been effectively used to manage hematopoietic malignancies and immunodeficiency. Despite the successful use several challenges remain. For autologous transplants, HSCs are routinely isolated from the peripheral blood following mobilization with G-CSF, however many patients that have been treated with chemotherapy are refractory to mobilization. In the allogeneic transplant setting, treatment related toxicity including graft vs. host disease, delayed or failed engraftment, and lack of suitable HLA-matched donors represent major challenges. Umbilical cord blood (CB) cells have great potential as an alternative source of HSCs for individuals who lack a HLA-matched donor, but at present have limited utility because of low HSC numbers per graft leading to delayed recovery. Ex-vivo expansion of HSCs is an attractive strategy to optimize autologous and allogeneic transplantation as engraftment speed (absolute neutrophil count &gt;500/μl) and success correlates positively with HSC dose. For this reason ex-vivo HSC expansion has been a subject of intense research for the past 20 years; however, identification of culture conditions that allow HSC expansion and long-term hematopoietic reconstitution have remained elusive. Recently, several groups have reported that signals other than hematopoietic growth factors, including ligands for G protein-coupled receptors and signaling molecules sensing neighboring cells such as notch may be required for optimal HSC expansion. Manipulation of signaling pathways using low molecular weight (LMW) compounds represents an alternative approach that can be exploited to regulate ex-vivo HSC expansion. To identify such compounds, we developed and performed an unbiased high-throughput screen for small molecules that regulate HSC self-renewal. The assay took advantage of advances in screening technology developed at GNF that permit low volume (10uL) screens to be conducted in massively parallel fashion using advanced automation and imaging technologies. These advances allow screens to be conducted on purified human CD34+ HSCs isolated from normal donors and circumvent a major limitation of the field- a lack of a suitable cell line model for human HSCs. From this screen we identified a small molecule (SR1) that regulates HSC self-renewal. Mobilized peripheral blood (mPB) CD34+ HSCs cultured with SR1 for 14 days had a ten-fold increase in the number of CD34+ cells compared to cultures without compound. The expansion of mPB CD34+ cells with SR1 for 1 week was associated with increased numbers of mixed (GM and GEMM) colony forming cells (CFC) and a 9-fold increase in the number of 4 week cobblestone area forming cells (CAFC). In the NOD-SCID repopulation assay, mPB CD34+ cells expanded with SR1 for 4 days displayed &gt;2-fold higher levels of engraftment compared to control cultures and uncultured cells. These data suggest that SR1 promotes the net expansion of NOD-SCID repopulating cells. To explore the utility of SR1 for expansion of CB HSC, CD34+ cells were isolated from CB and cultured in the presence or absence of SR1 for up to five weeks. Remarkably, SR1 supported the sustained growth of CB HSCs with &gt;100-fold increased numbers of CD34+ cells and CD34+CD133+CD38− cells compared to control cultures (Figure 1). In vitro assays of cord blood CD34+ cells expanded for 5 weeks with SR1 showed a 65-fold increase in total CFC and &gt;1000-fold increase in the number of GEMM CFC compared to control cultures. NOD-SCID repopulating experiments of expanded cord blood HSC are in progress. These results demonstrate that high throughput screening of LMW compound libraries is a viable approach to find novel regulators of HSC self-renewal and identify a compound class that greatly facilitates ex-vivo expansion of HSCs. Fig. 1 SR promotes sustained expansion of CB CD34 133+ CD38− cells Fig. 1. SR promotes sustained expansion of CB CD34 133+ CD38− cells

scholarly journals A Stemness Screen Reveals C3ORF54/INKA1 As a Gate-Keeper of Human Stem Cell Latency

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 325-325
Author(s):  
Kerstin B. Kaufmann ◽  
Laura Garcia Prat ◽  
Shin-Ichiro Takayanagi ◽  
Jessica McLeod ◽  
Olga I. Gan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Fold Increase ◽  
Steady Increase ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The controversy generated from recent murine studies as to whether hematopoietic stem cells (HSC) contribute to steady-state hematopoiesis emphasizes how limited our knowledge is of the mechanisms governing HSC self-renewal, activation and latency; a problem most acute in the study of human HSC and leukemia stem cells (LSC). Many hallmark stem cell properties are shared by HSC and LSC and therefore a better understanding of stemness regulation is crucial to improved HSC therapies and leukemia treatments targeting LSC. Our previous work on LSC subsets from >80 AML patient samples revealed that HSC and LSC share a transcriptional network that represent the core elements of stemness (Eppert, Nature Med 2011; Ng, Nature 2016). Hence, to identify the key regulators of LSC/HSC self-renewal and persistence we selected 64 candidate genes based on expression in functionally validated LSC vs. non-LSC fractions and assessed their potential to enhance self-renewal in a competitive in vivo screen. Here, we transduced cord blood CD34+CD38- cells with 64 barcoded lentiviral vectors to assemble 16 pools, each consisting of 8 individual gene-transduced populations, for transplantation into NSG mice. Strikingly, individual overexpression (OE) of 5 high scoring candidates revealed delayed repopulation kinetics of human HSC/progenitor cells (HSPC): gene-marking of human CD45+ and lin-CD34+ cells was reduced relative to input and control at 4w post transplantation, whereas by 20w engraftment of marked cells reached or exceeded input levels. For one of these candidates, C3ORF54/INKA1, we found that OE did not alter lineage composition neither in in vitro nor in vivo assays but increased the proportion of primitive CD34+ cells at 20w in vivo; moreover, secondary transplantation revealed a 4.5-fold increase in HSC frequency. Of note, serial transplantation from earlier time points (2w, 4w) revealed superior engraftment and hence greater self-renewal capacity upon INKA1-OE. Since we observed a 4-fold increase of phenotypic multipotent progenitors (MPP) relative to HSC within the CD34+ compartment (20w) we assessed whether INKA1-OE acts selectively on either cell population. The observation of latency in engraftment was recapitulated with sorted INKA1-OE HSC but not MPP. Likewise, liquid culture of HSPC and CFU-C assays on sorted HSC showed an initial delay in activation and colony formation upon INKA1-OE that was completely restored by extended culture and secondary CFU-C, respectively. INKA1-OE MPP showed a slight increase in total colony count in primary CFU-C and increased CDK6 levels in contrast to reduced CDK6 levels in INKA1-OE HSC emphasizing opposing effects of INKA1 on cell cycle entry and progression in either population. Taken together, this suggests that INKA1-OE preserves self-renewal capacity by retaining HSC preferentially in a latent state, however, upon transition to MPP leads to enhanced activation. Whilst INKA1 has been described as an inhibitor of p21(Cdc42/Rac)-activated kinase 4 (PAK4), no role for PAK4 is described in hematopoiesis. Nonetheless, its regulator Cdc42 is implicated in aging of murine HSPC by affecting H4K16 acetylation (H4K16ac) levels and polarity and has recently been described to regulate AML cell polarity and division symmetry. In our experiments immunostaining of HSPC subsets cultured in vitro and from xenografts indicates that INKA1-OE differentially affects epigenetics of these subsets linking H4K16ac to the regulation of stem cell latency. In AML, transcriptional upregulation of INKA1 in LSC vs. non-LSC fractions and at relapse in paired diagnosis-relapse analysis (Shlush, Nature 2017) implicates INKA1 as a regulator of LSC self-renewal and persistence. Indeed, INKA1-OE in cells derived from a primary human AML sample (8227) with a phenotypic and functional hierarchy (Lechman, Cancer Cell 2016) revealed a strong latency phenotype: In vitro and in vivo we observed label retention along with a steady increase in percentage of CD34+ cells, transient differentiation block, reduced growth rate, G0 accumulation and global reduction of H4K16ac. In summary, our data implicates INKA1 as a gate-keeper of stem cell latency in normal human hematopoiesis and leukemia. Studying the detailed pathways involved will shed light upon the mechanisms involved in HSC activation and latency induction and will help to harness these for novel therapeutic approaches. Disclosures Takayanagi: Kyowa Hakko Kirin Co., Ltd.: Employment.

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