High-Density Clonal Analysis Reveals Highly Active Contribution of Multipotent Hematopoietic Stem Cells during Early Phases of Hematopoietic Recovery after Transplantation

Abstract Recovery after conditioning and transplantation of hematopoietic stem and progenitor cells (HSPC) is thought to be biphasic, with short-term engrafting progenitors driving the recovery for 6-9 months and multipotent hematopoietic stem cells (HSCs) providing long-term repopulation. Recent clonal tracking data from autologous human gene therapy trials seems to support this model (Biasco et al. 2016, Cell Stem Cell; Six et al. 2020, Blood). These recent reports investigating the contribution of HSCs in patients are based on the longitudinal tracking of thousands of gene-marked cells using retroviral integration site analysis (ISA). While this technology is very reliable to follow gene therapy patients and monitor the potential outgrowth of dominant or malignant clones, low sensitivity and high error rates require significant data exclusion and sophisticated statistical tests to ensure data reliability (Adair et al. 2020, Molecular Therapy MCD). Lack of sensitivity can be overcome by increasing the frequency (high density) of sampling. However, limited material from patients remains a bottleneck for improved data quality and, consequently, correct interpretation of such complex datasets. To overcome the limitations of ISA and determine the onset of HSC contribution we performed high-density sampling for ISA in nonhuman primates (NHPs) transplanted with gene-modified HSCs. In the first month of hematopoietic recovery weekly blood samples were taken to enhance data density and increase the reliablity to detect clones with low abundance. Animals were followed up to 5 years to confirm that identified HSC clones persist long-term. Finally, clonal tracking data from the NHPs was used to inform a simulation of hematopoietic reconstitution, determine the temporal involvement of HSCs, and refine the phases of hematopoietic recovery after myeloablation and HSC transplantation. In contrast to the current biphasic model, contribution of multipotent HSCs clones was detected in the very first blood samples taken 2 to 3 weeks post-transplant during neutrophil recovery. HSC clones found in these early time points persisted long-term throughout the entire follow-up and were detected in bone marrow CD34 + cells 4 years later. Most surprisingly, multipotent HSCs became the dominant source for mature blood cells in the peripheral blood as early as 50 days post-transplant. To understand the observed kinetics of HSC contribution and change in clonal diversity in our dataset, we simulated the clonal outgrowth and differentiation of multipotent clones. Simulations predicted that hematopoietic recovery is primarily HSC driven and HSC contribution follows a stochastic pattern. Finally, to confirm that HSCs proliferation and differentiation is a stochastic process, in vitro experiments in colony-forming cell (CFC) assays were carried out. As predicted, the decision of individual HSCs to either grow into a larger pool or differentiate and get lost followed the same kinetics as observed in vivo. Here, we show evidence that long-term persisting multipotent HSCs actively contribute during early hematopoietic reconstitution after myeloablation and HSC transplantation. Enhanced sampling showed that multipotent HSCs produce neutrophils during recovery and become the predominant source of mature blood cells as early as 50 days post-transplant. Most importantly, observed changes in the clonal diversity during early recovery suggest a stochastic engraftment of HSCs rather than a bi-phasic reconstitution initially driven by short-term progenitors. These findings should have important implications for the design of ex vivo and in vivo HSC gene therapy and genome editing approaches. Figure 1 Figure 1. Disclosures Radtke: 47 Inc.: Consultancy; Ensoma Inc.: Consultancy. Kiem: Homology Medicines: Consultancy; VOR Biopharma: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company.

Download Full-text

Hematopoietic recovery after transplantation is primarily derived from the stochastic contribution of hematopoietic stem cells

10.1101/2021.09.21.461235 ◽

2021 ◽

Author(s):

Stefan Radtke ◽

Mark Enstrom ◽

Dnyanada P. Pande ◽

Margaret L. Cui ◽

Ravishankar Madhu ◽

...

Keyword(s):

Clonal Diversity ◽

Enhanced Sampling ◽

Primate Model ◽

Hematopoietic Stem ◽

Neutrophil Recovery ◽

Hematopoietic Recovery ◽

Hematopoietic Reconstitution ◽

Initial Recovery ◽

Two Phases

Reconstitution after hematopoietic stem cell (HSC) transplantation is assumed to occur in two distinct phases: initial recovery mediated by short-term progenitors and long-term repopulation by multipotent HSCs which do not contribute to hematopoietic reconstitution during the first 6-9 months. We have previously reported the transplantation and exclusive engraftment of the HSC-enriched CD34+CD45RA-CD90+ phenotype in a nonhuman primate model. Here, we closely followed the clonal diversity and kinetics in these animals. Enhanced sampling and high density clonal tracking within the first 3 month revealed that multipotent HSCs actively contributed to the early phases of neutrophil recovery and became the dominant source for blood cells as early as 50 days after transplant. Longitudinal changes in clonal diversity supported a stochastic engraftment of HSCs with the majority of HSCs clones vanishing early during neutrophil recovery and a smaller fraction of HSC clones expanding into bigger pools to support long-term hematopoiesis. In contrast to the bi-phasic model, we propose that hematopoietic recovery after myeloablation and transplantation is primarily derived from HSCs in a stochastic manner rather than in two phases by independent cell populations.

Download Full-text

Persistence of CRISPR/Cas9-Edited Hematopoietic Stem and Progenitor Cells and Reactivation of Fetal Hemoglobin in Nonhuman Primates

Blood ◽

10.1182/blood-2018-99-112996 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 806-806

Author(s):

Olivier Humbert ◽

Stefan Radtke ◽

Ray R Carillo ◽

Anai M Perez ◽

Sowmya Somashekar Reddy ◽

...

Keyword(s):

Gene Therapy ◽

Progenitor Cells ◽

Fetal Hemoglobin ◽

Hematopoietic Stem ◽

Hematopoietic Recovery ◽

Stem And Progenitor Cells ◽

Therapy Model ◽

Equity Ownership

Abstract Beta-thalassemia and sickle cell disease are monogenic disorders that are currently treated by allogeneic bone marrow (BM) transplantation although the challenges of finding a suitable matched-donor and the risk of graft vs host disease have limited the adoption of this otherwise curative treatment. A potentially promising approach for hemoglobinopathies aims to reactivate fetal hemoglobin (HbF) as a substitute for defective or absent adult hemoglobin by modifying the patient's own hematopoietic stem and progenitor cells (HSPCs). Here, we evaluated CRISPR/Cas9-induced small deletions in HSPCs that are associated with hereditary persistence of fetal hemoglobin (HPFH) using our nonhuman primate (NHP) stem cell transplantation and gene therapy model. The CRISPR/Cas9 nuclease platform was employed to recapitulate a natural genetic alteration identified in individuals with HPFH, consisting of a 13-nucleotide (nt) deletion in the gamma globin gene promoter. A first cohort of three rhesus macaques received 70-75% HPFH-edited BM-derived CD34+ HSPCs. All animals showed rapid hematopoietic recovery and peripheral blood (PB) editing levels stabilized at 12-30% for at least a year post transplantation (Figure 1). HbF production, determined by circulating F-cells, persisted at frequencies of 8-22% and correlated with in vivo PB editing. Robust engraftment of gene-edited HSPCs in the BM compartment was observed in all animals, with no measurable off-target activity or clonal expansion. We have recently shown, that the CD34+CD90+CD45RA- phenotype is exclusively required for short- and long-term multilineage reconstitution, significantly reduces the target cell number for gene therapy/editing and is conserved between human and NHP hematopoietic cells (Radtke et al., STM, 2017). To explore this cell population further, we transplanted a second cohort of three animals by sort-purifying and solely editing this hematopoietic stem cell (HSC)-enriched CD34+CD90+CD45RA- phenotype, thus reducing the number of target cells by over 10-fold without impacting hematopoietic recovery, engraftment, or HbF reactivation. In vivo levels of gene-edited PB started at less than 5% because of the high number of co-infused unmodified progenitor cells, but rapidly increased to about 50% within 1 week (Figure 1) and stabilized at levels comparable to the CD34 cohort. This data supports our interpretation that CD34+CD90+CD45RA- cells are the main cell population relevant for long-term reconstitution and an excellent target for improved and efficient gene therapy/editing. These results demonstrate robust engraftment and persistence of CD34+ HPSCs as well as HSC-enriched CD34+CD90+CD45RA- cells that have been CRISPR/Cas9-edited at the 13nt-HPFH site, with marked and stable HbF reactivation and no overt adverse effects in a NHP transplantation and gene therapy model. Most importantly, we validated our refined CD90+ target which reduces the need for editing reagents by 90% without compromising the gene modification and engraftment efficiencies. These are the first data in a clinically relevant large animal model to demonstrate the feasibility and clinical applicability of CRISPR/Cas9-mediated fetal hemoglobin reactivation. The successful targeting and engraftment of our HSC-enriched population should also have significant implications for gene therapy and editing of other genetic diseases. Figure 1: Tracking of HPFH editing in transplanted animals. A) Editing efficiency was longitudinally determined by next generation sequencing of the targeted locus in PB white blood cells from 2 cohorts of transplanted rhesus animals. Frequency is represented as the proportion of all sequence reads containing an edited locus. B) Normalized frequency of the desired 13nt-HPFH deletion in the same animals as shown in A). Figure. Figure. Disclosures Negre: Bluebird Bio: Employment, Equity Ownership, Other: Salary. Adair:RX Partners: Honoraria; Miltenyi Biotec: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy; Magenta: Consultancy; Homology Medicine: Consultancy.

Download Full-text

CD26 Inhibitor Treated and CD26−/− Recipient Mice Exhibit Improved Transplant Efficiency as Quantitated by Increased Short-Term Homing and Long-Term Engraftment

Blood ◽

10.1182/blood.v112.11.361.361 ◽

2008 ◽

Vol 112 (11) ◽

pp. 361-361 ◽

Cited By ~ 1

Author(s):

Laura A. Paganessi ◽

Stephanie A. Gregory ◽

Henry C. Fung ◽

Kent W. Christopherson

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mononuclear Cells ◽

Short Term ◽

Hematopoietic Stem ◽

Post Transplant ◽

Inhibitor Treatment

Abstract A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/ HPC) trafficking is believed to be critical for the development of methodologies to improve transplant efficiency and subsequently immune reconstitution during hematopoietic stem cell transplantation in the clinical setting. Through the use of CD26 inhibitors and CD26 deficient mice (CD26−/−), we have previously generated data in mice suggesting that suppression of CD26/DPPIV (dipeptidylpeptidase IV) enzymatic activity on the transplant donor cell population can be utilized as a method of increasing transplant efficiency (Christopherson, KW 2nd, et al, Science 2004. 305:1000–3). However, the clinical importance of the transplant recipient should not to be overlooked given the potential importance of the bone marrow microenvironment in regulating the transplant process. We therefore investigated here whether inhibition or loss of CD26 activity in recipient mice would have an effect on transplant efficiency utilizing an in vivo congenic mouse model of transplantation. The short-term homing and long-term engraftment of BoyJ donor cells (expressing CD45.1+) into lethally irradiated control C57BL/6, CD26 inhibitor (Diprotin A) treated C57BL/6, or CD26−/− mice (expressing CD45.2+) was monitored by flow cytometric analysis of the bone marrow and peripheral blood at 24 hours and 6 months post-transplant respectively. Twenty-four hours post-transplant of 20×106 BoyJ mononuclear cells, we observed 8.85±0.58%, 10.69±1.01%, and 12.45±1.33% donor derived Sca-1+lin− cells in the bone marrow of recipient mice for control, Diprotin A treated, and CD26−/− recipient mice respectively. As compared to control mice, this represents a 20.8% increase (p=0.01) with CD26 inhibitor treatment and a 40.7% increase (p£0.05) resulting from the use of a CD26−/− recipient in short-term homing (N=5 mice per group). Six months post-transplant of 1×105 BoyJ mononuclear cells, we observed 39.90± 4.38%, 70.22± 3.72%, and 92.51± 1.04% donor contribution to hematopoiesis in the peripheral blood of control, Diprotin A treated, and CD26−/− recipient mice respectively. This represents a 76.0% increase (p£0.01) with CD26 inhibitor treatment and a 131.9% increase (p£0.01) as a result of the CD26−/− recipient in long-term engraftment as compared to control recipient mice (N=14 mice per group). These results provide pre-clinical evidence of the importance of CD26 expression within the transplant recipient with regard to regulating hematopoietic stem cell homing and engraftment. Our results also support the potential use of CD26 inhibitors to treat transplant patients during hematopoietic stem cell transplantation as a method of improving transplant efficiency. Lastly, our use of inhibitor treated C57BL/6 and CD26−/− recipient mice, which are also on a C57BL/6 background, in conjunction with a congenic model of transplantation provides a accurate and convenient model system for the in vivo testing of the efficacy of existing and new CD26 inhibitors in transplant recipients.

Download Full-text

Systemic Administration of Pleiotrophin Induces Hematopoietic Stem Cell Regeneration In Vivo.

Blood ◽

10.1182/blood.v114.22.1498.1498 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1498-1498

Author(s):

Heather A Himburg ◽

Pamela Daher ◽

Sarah Kristen Meadows ◽

J. Lauren Russell ◽

Phuong Doan ◽

...

Keyword(s):

Stem Cell ◽

Growth Factor ◽

Progenitor Cells ◽

Progenitor Cell ◽

Fold Increase ◽

Cell Regeneration ◽

Hematopoietic Stem ◽

Hematopoietic Reconstitution

Abstract Abstract 1498 Poster Board I-521 Significant progress has been made toward delineating the intrinsic and extrinsic signaling pathways that regulate hematopoietic stem cell (HSC) self-renewal. However, much less is known regarding the process of HSC regeneration or the extrinsic signals that regulate hematopoietic reconstitution following stress or injury. Elucidation of the microenvironmental signals which promote HSC regeneration in vivo would have important implications for the treatment of patients undergoing radiation therapy, chemotherapy and stem cell transplantation. We recently reported that pleiotrophin, a soluble heparin-binding growth factor, induced a 10-fold expansion of murine long-term repopulating HSCs in short term culture (Himburg et al. Blood (ASH Annual Meeting Abstracts), Nov 2008; 112: 78). Based on this observation, we hypothesized that PTN might also be a regenerative growth factor for HSCs. Here we tested the effect of systemic administration of PTN to non-irradiated and irradiated C57Bl6 mice to determine if PTN could promote HSC regeneration in vivo. C57Bl6 mice were irradiated with 700 cGy total body irradiation (TBI) followed by intraperitoneal administration of 2 μg PTN or saline x 7 days, followed by analysis of BM stem and progenitor cell content. Saline-treated mice demonstrated significant reductions in total BM cells, BM c-kit+sca-1+lin- (KSL) cells, colony forming cells (CFCs) and long term culture-initiating cells (LTC-ICs) compared to non-irradiated control mice. In contrast, PTN-treated mice demonstrated a 2.3-fold increase in total BM cells (p=0.03), a 5.6-fold increase in BM KSL stem/progenitor cells (p=0.04), a 2.9-fold increase in BM CFCs (p=0.004) and an 11-fold increase in LTC-ICs (p=0.03) compared to saline-treated mice. Moreover, competitive repopulating transplantation assays demonstrated that BM from PTN-treated, irradiated mice contained 5-fold increased competitive repopulating units (CRUs) compared to saline-treated, irradiated mice (p=0.04). Taken together, these data demonstrate that the administration of PTN induces BM HSC and progenitor cell regeneration in vivo following injury. Comparable increases in total BM cells, BM KSL cells and BM CFCs were also observed in PTN-treated mice compared to saline-treated controls following 300 cGy TBI, demonstrating that PTN is a potent growth factor for hematopoietic stem/progenitor cells in vivo at less than ablative doses of TBI. In order to determine whether PTN acted directly on BM HSCs to induce their proliferation and expansion in vivo, we exposed mice to BrDU in their drinking water x 7 days and compared the response to saline treatment versus PTN treatment. PTN-treated mice demonstrated a significant increase in BrDU+ BM KSL cells compared to saline-treated controls (p=0.04) and cell cycle analysis confirmed a significant increase in BM KSL cells in S phase in the PTN-treatment group compared to saline-treated controls (p=0.04). These data indicate that PTN serves as a soluble growth factor for BM HSCs and induces their proliferation and expansion in vivo while preserving their repopulating capacity. These results suggest that PTN has therapeutic potential as a novel growth factor to accelerate hematopoietic reconstitution in patients undergoing myelosuppressive radiotherapy or chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

In Vivo Selection Unmasks a Dormant Pool of Repopulating Hematopoietic Clones

Blood ◽

10.1182/blood.v126.23.242.242 ◽

2015 ◽

Vol 126 (23) ◽

pp. 242-242

Author(s):

Jennifer E Adair ◽

Lauren E Schefter ◽

Daniel R Humphrys ◽

Kevin G Haworth ◽

Jonah D Hocum ◽

...

Keyword(s):

Conflicts Of Interest ◽

Selective Advantage ◽

First Year ◽

Short Term ◽

Hematopoietic Stem ◽

In Vivo Selection ◽

Chemotherapy Administration ◽

Cd34 Cells

Abstract Long-term clonal tracking studies utilizing hematopoietic stem and progenitor cells (HSPCs) in nonhuman primates receiving myeloablative transplantation demonstrate a successive pattern of repopulation: short-term repopulating cells are succeeded by long-term clones. However, the duration of short-term repopulation and the numbers of clones contributing to either short or long-term repopulation are unclear. Here, we tracked >11,000 unique clones in 8 pigtail macaques for up to 9 years following myeloablative transplantation with autologous, lentivirus gene-modified CD34+ HSPCs. Seven of these animals received cells expressing the P140K mutant methylguanine methyltransferase transgene, which is resistant to the combination of O6-benzylguanine (O6BG) and bis-chloroethylnitrosourea (BCNU) chemotherapy, thus conferring a selective advantage to gene-modified cells in vivo. After transplantation and before in vivo selection with O6BG/BCNU, we observed a successive pattern of hematopoietic reconstitution, with short-term clones declining within 100 days after transplantation. Within the first year after transplant, the percent of persistent clones varied from animal-to-animal, ranging from 8% to 54% of clones detected at a >1% frequency, and remained stable in the absence of selective pressure. Importantly, when animals engrafted with P140K-expressing cells were administered O6BG/BCNU we observed novel clonal patterns, which directly correlated with transplanted cell dose and time of chemotherapy administration after transplant. In all animals, chemotherapy induced emergence of previously undetected clones. In animals receiving ≤12x106 CD34+ cells/kg at the time of transplant (n = 4), chemotherapy also induced a re-emergence of previously declined short-term repopulating clones or a stabilization (i.e. decreased fluctuation) of repopulating clones identified between 100 days and 1 year after transplant. However, in animals receiving robust cell doses, ≥35x106 CD34+ cells/kg (n = 2), chemotherapy more than 1 year after transplant induced a completely novel clonal repertoire. In one animal receiving 22x106 CD34+ cells/kg at transplant, chemotherapy administration beginning <1 year (253 days) after transplant induced clonal stability, which was maintained through two additional chemotherapy treatments. These data suggest that some short-term repopulating clones may have long-term repopulation ability, but revert to a dormant phase within the first year after transplant. Additionally, these data indicate that transplant of excess repopulating cells results in early dormancy of a large proportion of repopulating clones. Together, these findings suggest that previous estimates of HSPC frequency based on clone tracking are an underestimate of true graft repopulation potential. Disclosures No relevant conflicts of interest to declare.

Download Full-text

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

Blood ◽

10.1182/blood.v90.11.4354 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4354-4362 ◽

Cited By ~ 25

Author(s):

Nobuko Uchida ◽

Annabelle M. Friera ◽

Dongping He ◽

Michael J. Reitsma ◽

Ann S. Tsukamoto ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Cell Number ◽

Short Term ◽

Synthesis Inhibitor ◽

Hematopoietic Stem ◽

Cell Cycle Status

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

Download Full-text

The HGFIN Gene Mediates Cell Cycle Quiescence of CD34+/CD38-: Implications for Hematopoietic Stem Cell Expansion and Gene Therapy.

Blood ◽

10.1182/blood.v104.11.1698.1698 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1698-1698

Author(s):

Era Ahmeti ◽

Junichi Sadoshima ◽

Jonathan S. Harrison ◽

Pranela Rameshwar

Keyword(s):

Cell Cycle ◽

Gene Therapy ◽

Cell Cycle Progression ◽

Transmembrane Protein ◽

Short Term ◽

Hematopoietic Stem ◽

G0 Phase ◽

Expansion Media

Abstract Hematopoietic stem cells (HSC) are difficult to expand in vitro since expansion media mostly include exogenous factor, e.g., cytokines. Growth factors cause lineage commitment with the HSC exhibiting short-term immune reconstitution. The recently cloned HGFIN (or nmb) gene has been detected in CD34+/CD38− and differentiated immune cells. HGFIN is a single transmembrane protein with multiple consensus regions for p53. In malignant cells, HGFIN mediate tumor progression and in normal cells, HGFIN maintain cell cycle quiescence. We hypothesize that HGFIN negatively regulates proliferation of HSC by prolonging the G0 phase. Short-term knockdown of HGFIN in HSC could cause the cells to exit G0/G1 phase so as to accommodate the insertion of a replacement gene, without changing the pluripotent property of HSC. RT-PCR detected HGFIN mRNA in CD34+/CD38− cells, but not in CD34+/CD38+ cells from bone marrow (BM) aspirates. Treatment of CD34+/CD38− cells with double stranded HGFIN siRNA oligos for 2 h led to their exit from G1 to G2/S (flow cytometry). During this time, adenovirus-LacZ (ß-galactosidase assay) was incorporated in >80% CD34+/CD38− cells, compared to 10–20% exposed to mutant siRNA, or untreated. After 24 h exposure to siRNA oligos, the CD34+/CD38− cells reverted to G0/1 phase. The latter cells (with adenovirus-LacZ) were studied in 12-wk long-term culture initiating assay. The results (ß-gal positive progenies) from the LTC-IC assay indicate that despite siRNA treatment, the CD34+/CD38− cells retained multipotential properties. Future investigations with specific HGFIN antibody will map pathways among HGFIN and other molecules that regulate cell cycle progression in CD34+/CD38− cells. In summary, the results show that HGFIN regulate cell cycle quiescence of CD34+/CD38− cells. Transient knock down of HGFIN in these cells allow adenovirus to be inserted without loss of multipotential properties. Understanding the biology of HSC at the molecular level will be relevant to HSC expansion and restoration of immune competence via gene therapy.

Download Full-text

Notch2 Signaling Inhibits Differentiation and Promotes Self Renewal of Hematopoietic Stem and Progenitor Cells During Marrow Regeneration.

Blood ◽

10.1182/blood.v114.22.2544.2544 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2544-2544

Author(s):

Barbara Varnum-Finney ◽

Irwin D. Bernstein

Keyword(s):

Progenitor Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Myeloid Differentiation ◽

Primary Control ◽

Short Term ◽

Self Renewal ◽

Hematopoietic Stem ◽

Post Transplant

Abstract Abstract 2544 Poster Board II-521 Notch regulates numerous lineage choices during vertebrate development, and although ex vivo studies suggest that Notch regulates hematopoietic stem cell (HSC) and multipotential progenitor (MPP) differentiation, a functional role for Notch in HSC/MPP self renewal in vivo remains controversial. We previously reported a Notch2 signaling role during bone marrow (BM) recovery following injection with chemotherapeutic agent 5-fluorouracil (5FU), where Notch2 signaling impedes myeloid differentiation, allowing for generation of sufficient numbers of progenitor cells. Herein, we examine a Notch2 signaling role in HSC as well as progenitor cell self renewal by enumerating generation of HSC and short term repopulating cells in lethally irradiated recipients (Ly5.1+) transplanted with a limiting number (5 × 105) of BM cells from either control mice or from mice bearing Cre-LoxP-inducible Notch2 deletions (Ly5.2+). In recipient mice transplanted with control BM, recovery was evident from Day11 to Day13 post transplant when significantly more than the initial post-irradiation number of 9.0 × 106 BM cells was seen in the recovering marrow. In recovering mice, recipients receiving control cells generated more BM cells than did recipients receiving Notch2-deficient cells. Furthermore, mice receiving control cells generated significantly more donor Sca-1+c-kit+ (SK+) cells than recipients receiving Notch2-deficient BM cells [44.4×103 (s.e.m.+/− 14×103) vs 8.2×103 (s.e.m.+/−1.5×103), respectively, p=0.001]. To quantitate the generation of short term repopulating cells, secondary radioprotection assays were performed. Irradiated secondary recipient mice received 1×106 BM cells from the primary recipients previously transplanted with either control cells or Notch2-deficient cells. Secondary recipients receiving cells from primary control transplants survived significantly longer than those receiving cells from primary Notch2-deficient transplants or than irradiated mice receiving no cells (n=4, p=0.01), indicating Notch2 is required to generate sufficient numbers of cells to provide radioprotection. To quantitate long term HSC generated in the recovering marrow, competitive repopulating units (CRU) were enumerated by performing secondary transplants in which 4-doses of BM cells ranging from 4 × 104 to 5 × 106 cells from primary transplants were injected into secondary recipients along with 1 × 105 Ly5.1+ competing cells. Enumeration of CRU at 2 weeks post transplant confirmed the number of short term repopulating cells was significantly decreased in mice transplanted with Notch2-deficient cells compared to mice transplanted with control cells [(1.3 CRU vs 8.8 CRU / 1×106 BM cells, respectively), p=0.0004)]. Enumeration of CRU at 9 weeks post transplant indicated HSC numbers were also significantly decreased in mice transplanted with Notch2-deficient cells compared to mice transplanted with control cells [(0.1 CRU vs 0.7 CRU / 1×106 BM cells, respectively), p=0.02]. Taken together, our results demonstrate a role for Notch2 in enhancing generation of long term HSC as well as short term repopulating cells and suggests that Notch2 signaling regulates a hierarchy of events to assure the initial repopulation by HSC and MPP, while delaying myeloid differentiation during hematopoietic regeneration. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

In Vivo Modulation of Mitochondrial Activity Determines HSC Engraftment and Post-Transplant Survival in Mice

Blood ◽

10.1182/blood.v120.21.213.213 ◽

2012 ◽

Vol 120 (21) ◽

pp. 213-213

Author(s):

Nicola Vannini ◽

Olaia M. Naveiras ◽

Vasco Campos ◽

Eija Pirinen ◽

Riekelt Houtkooper ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

High Fat Diet ◽

Mitochondrial Activity ◽

High Fat ◽

Short Term ◽

Hematopoietic Stem ◽

Post Transplant ◽

Progenitor Compartment

Abstract Abstract 213 Cellular metabolism is emerging as a potential fate determinant in cancer and stem cell biology, constituting a crucial regulator of the hematopoietic stem cell (HSC) pool [1–4]. The extremely low oxygen tension in the HSC microenvironment of the adult bone marrow forces HSCs into a low metabolic profile that is thought to enable their maintenance by protecting them from reactive oxygen species (ROS). Although HSC quiescence has for long been associated with low mitochondrial activity, as testified by the low rhodamine stain that marks primitive HSCs, we hypothesized that mitochondrial activation could be an HSC fate determinant in its own right. We thus set to investigate the implications of pharmacologically modulating mitochondrial activity during bone marrow transplantation, and have found that forcing mitochondrial activation in the post-transplant period dramatically increases survival. Specifically, we examined the mitochondrial content and activation profile of each murine hematopoietic stem and progenitor compartment. Long-term-HSCs (LT-HSC, Lin-cKit+Sca1+ (LKS) CD150+CD34-), short-term-HSCs (ST-HSC, LKS+150+34+), multipotent progenitors (MPPs, LKS+150-) and committed progenitors (PROG, Lin-cKit+Sca1-) display distinct mitochondrial profiles, with both mitochondrial content and activity increasing with differentiation. Indeed, we found that overall function of the hematopoietic progenitor and stem cell compartment can be resolved by mitochondrial activity alone, as illustrated by the fact that low mitochondrial activity LKS cells (TMRM low) can provide efficient long-term engraftment, while high mitochondrial activity LKS cells (TMRM high) cannot engraft in lethally irradiated mice. Moreover, low mitochondrial activity can equally predict efficiency of engraftment within the LT-HSC and ST-HSC compartments, opening the field to a novel method of discriminating a population of transitioning ST-HSCs that retain long-term engraftment capacity. Based on previous experience that a high-fat bone marrow microenvironment depletes short-term hematopoietic progenitors while conserving their long-term counterparts [5], we set to measure HSC mitochondrial activation in high-fat diet fed mice, known to decrease metabolic rate on a per cell basis through excess insulin/IGF-1 production. Congruently, we found lower mitochondrial activation as assessed by flow cytometry and RT-PCR analysis as well as a depletion of the short-term progenitor compartment in high fat versus control chow diet fed mice. We then tested the effects of a mitochondrial activator known to counteract the negative effects of high fat diet. We first analyzed the in vitro effect on HSC cell cycle kinetics, where no significant change in proliferation or division time was found. However, HSCs responded to the mitochondrial activator by increasing asynchrony, a behavior that is thought to directly correlate with asymmetric division [6]. As opposed to high-fat diet fed mice, mice fed with the mitochondrial activator showed an increase in ST-HSCs, while all the other hematopoietic compartments were comparable to mice fed on control diet. Given the dependency on short-term progenitors to rapidly reconstitute hematopoiesis following bone marrow transplantation, we tested the effect of pharmacological mitochondrial activation on the recovery of mice transplanted with a limiting HSC dose. Survival 3 weeks post-transplant was 80% in the treated group compared to 0% in the control group, as predicted by faster recovery of platelet and neutrophil counts. In conclusion, we have found that mitochondrial activation regulates the long-term to short-term HSC transition, unraveling mitochondrial modulation as a valuable drug target for post-transplant therapy. Identification of molecular pathways accountable for the metabolically mediated fate switch is currently ongoing. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

E47 regulates hematopoietic stem cell proliferation and energetics but not myeloid lineage restriction

Blood ◽

10.1182/blood-2010-07-297689 ◽

2011 ◽

Vol 117 (13) ◽

pp. 3529-3538 ◽

Cited By ~ 18

Author(s):

Qi Yang ◽

Brandt Esplin ◽

Lisa Borghesi

Keyword(s):

Energy Metabolism ◽

Myeloid Differentiation ◽

Short Term ◽

Hematopoietic Stem ◽

Altered Expression ◽

Helix Loop Helix ◽

Multipotent Progenitors

Abstract The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins, the widely expressed basic helix-loop-helix transcription factors, contribute to HSC and MPP activity, but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches, we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However, long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover, E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism, and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together, these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism, and demonstrate that E47 is not required for short-term myeloid differentiation.

Download Full-text