scholarly journals CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4136-4142 ◽  
Author(s):  
I Kawashima ◽  
ED Zanjani ◽  
G Almaida-Porada ◽  
AW Flake ◽  
H Zeng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
In Utero ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Show Evidence ◽  
Marrow Cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

CCR2 Mediates Hematopoietic Stem/Progenitor Cell Trafficking to Peripheral Tissues after Inflammation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 472-472
Author(s):  
Yue Si ◽  
Chia-Lin Tsou ◽  
Israel Charo
Keyword(s):  
Bone Marrow ◽  
Chemokine Receptor ◽  
Bone Marrow Cells ◽  
Cell Trafficking ◽  
Aseptic Inflammation ◽  
Hematopoietic Stem ◽  
Peripheral Tissues ◽  
Marrow Cells

Abstract Hematopoietic stem cells (HSCs) are bone-marrow derived, self-renewing pluripotent cells that give rise to terminally differentiated circulating blood cells. HSCs have been implicated in parenchymal tissue repair in the setting of inflammation. In response to the antagonist of the chemokine receptor CXCR4, HSCs and their progenitors migrate from bone marrow to the blood. However, little is known about the signals that mediate their trafficking from the blood into peripheral tissues. Recently, we showed that mice genetically deficient in chemokine receptor CCR2 (CCR2−/− mice) have a marked decrease in the number of circulating “inflammatory” (7/4+, Ly6c+) monocytes, but no decrease in myeloid progenitor cells in the bone marrow (Tsou et al, J Clin Invest, 2007, 902). These data indicated that although CCR2 is not necessary for HSCs to differentiate into mature monocytes, it does play a role in monocyte egress from bone marrow to blood. In the current study, we extend this work and investigate the expression of CCR2 on HSCs, and tested the hypothesis that CCR2 is critical for the recruitment of circulating HSCs to sites of inflammation. We found that CCR2 was expressed on subsets of primitive HSCs and myeloid progenitors and mediated HSC movement in response to inflammation. Using traditional transwell chambers, we found that c-Kit+Lin− cells derived from bone marrow underwent chemotaxis in response to the CCR2 ligands MCP-1 (CCL2) and MCP- 3 (CCL7). To determine whether CCR2 mediates HSC movement in vivo, we treated wildtype mice with thioglycollate to induce aseptic inflammation. HSCs were actively recruited to the peritoneum, as shown by fluorescence-activated cell sorting and functional colony formation assays. In contrast, this response was profoundly impaired in CCR2−/− mice. To determine whether the clonogenic cells recruited to peritoneum were true HSCs, we performed competitive transplantation assays. Thioglycollate was instilled into wildtype CD45.2+ mice, and peritoneal Lin− cells were collected, purified, and infused, together with CD45.1+ bone marrow cells, into lethally irradiated CD45.1+ mice. Four months later, up to 12% of the leukocytes in the peripheral blood of these primary recipient mice were CD45.2+. At the time of sacrifice, bone marrow cells were collected from these mice and injected into lethally irradiated secondary CD45.1+ recipient mice. Two months following the transplantation, up to 9% of the blood leukocytes in these secondary recipient mice were CD45.2+, confirming that long-term repopulating HSCs were recruited to the inflamed peritoneum of the donor mice. These findings suggest a novel role for CCR2 in the recruitment of long-term repopulating HSCs to sites of inflammation and injury. We are currently investigating whether recruited HSCs and their progenitors hasten the resolution of the inflammatory response or promote the repair of injured tissue.

scholarly journals Normal cycling patterns of hematopoietic stem cell subpopulations: an assay using long-term in vivo BrdU infusion

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1460-1462 ◽  
Author(s):  
ME Pietrzyk ◽  
GV Priestley ◽  
NS Wolf
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Improved Method ◽  
Hematopoietic Stem ◽  
Infusion Study ◽  
A Cell ◽  
Cell Subpopulations ◽  
Over Time

It was found in a long-term bromodeoxyuridine (BrdU) infusion study that two or more different subpopulations of bone marrow stem cells exist in mice. One of these subpopulations appears to be noncycling and forms approximately 10% of eight-day CFU-S. Another one, a subpopulation of slowly cycling bone marrow cells, is represented as 14- day CFU-S. The 14-day CFU-S have a regular increment in the percentage of the subpopulation entering the cycle over time, with a cell generation half-time of 21 days. The cycling status in these experiments was ascertained by in vivo continuous long-term BrdU infusion. An improved method is presented for long-term BrdU infusion with UV killing of cycled cells.

Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 700-704 ◽  
Author(s):  
Kimberly A. Gush ◽  
Kai-Ling Fu ◽  
Markus Grompe ◽  
Christopher E. Walsh
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mitomycin C ◽  
Fanconi Anemia ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Hematopoietic Stem ◽  
Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 829-836 ◽  
Author(s):  
Timothy W. Austin ◽  
Suzan Salimi ◽  
Gabor Veres ◽  
Franck Morel ◽  
Heini Ilves ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Marrow Cells

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR+ cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin−/loSca-1+ and c-kit+Sca-1+ subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR+c-kit+Sca-1+bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR+ cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.

scholarly journals Observations on the binding and interaction of radioiodinated colony- stimulating factor with murine bone marrow cells in vivo

Blood ◽  
1982 ◽  
Vol 59 (2) ◽  
pp. 408-420 ◽  
Author(s):  
G Pigoli ◽  
A Waheed ◽  
RK Shadduck
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Cell Interaction ◽  
Peritoneal Macrophages ◽  
Colony Stimulating Factor ◽  
Murine Bone Marrow ◽  
Stimulating Factor ◽  
Marrow Cells

Abstract Radioiodinated L-cell-derived colony-stimulating factor (CSF) was used to characterize the binding reaction to murine bone marrow cells. The major increment in cell-associated radioactivity occurred over 24 hr incubation at 37 degrees C, but virtually no binding was observed at 4 degrees C. The reaction was saturable with approximately 1 ng/ml of purified CSF. Unlabeled CSF prevented the binding, whereas a number of other hormones and proteins did not compete for CSF uptake. Further specificity studies showed virtually no binding to human bone marrow, which is unresponsive to this form of murine CSF. Minimal CSF uptake was noted with murine peritoneal macrophages, but virtually no binding was detected with thymic, lymph node, liver, or kidney cells. The marrow cell interaction with tracer appeared to require a new protein synthesis, as the binding was prevented by cycloheximide or puromycin. Preincubation of marrow cells in medium devoid of CSF increased the degree of binding after 1 hr exposure to the tracer. This suggests that CSF binding sites may be occupied or perhaps decreased in response to ambient levels of CSF in vivo. Approximately 70% of the bound radioactivity was detected in the cytoplasm at 24 hr. This material was partially degraded as judged by a decrease in molecular weight from approximately 62,000 to 2 peaks of approximately 32,000 and approximately 49,000, but 72% of the binding activity was retained. After plateau binding was achieved, greater than 80% of the radioactivity released into the medium was degraded into biologically inactive peptides with molecular weights less than 10,000. These findings suggest that the interaction of CSF with marrow cells is characterized by binding with subsequent internalization and metabolic degradation into portions of the molecule that are devoid of biologic activity.

Dynamic Patterns of Migration and Expansion of Hematopoiesis during MGMT Mediated Drug Selection.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 156-156 ◽  
Author(s):  
Yuan Lin ◽  
Perrin Cheung ◽  
David L. Wilson ◽  
Stanton L. Gerson
Keyword(s):  
Bone Marrow ◽  
In Vivo Imaging ◽  
Bone Marrow Cells ◽  
Clonal Expansion ◽  
Dependent Manner ◽  
Drug Selection ◽  
Hematopoietic Stem ◽  
Mouth Disease Virus ◽  
Marrow Cells

Abstract While hematopoietic engraftment kinetics are well appreciated after lethal irradiation in the mouse, most observations have been limited to blood samples or terminal examination of marrow or spleen. The development of non-invasive bioluminescence in vivo imaging technology allows a dynamic picture of engraftment and clonal expansion to be defined. We have extended this technology to the process of drug resistance gene therapy. We hypothesized that drug selection would profoundly affect the extent and dynamics of hematopoietic stem cells (HSC) engraftment and clonal expansion after lentiviral mediated gene transfer of the P140KMGMT gene into murine HSC. In previous studies, we have shown that P140KMGMT gene containing retroviral and lentiviral transduced bone marrow cells provided significant protection against chemotherapeutic drugs BCNU and TMZ given with BG (O6-Benzylguanine), in vitro and in vivo. We generated a bicistronic lentiviral vector containing P140KMGMT gene and firefly luciferase gene linked by 2A sequence of FMDV(Foot-and-Mouth Disease Virus), which will cleave itself during ribosomal translation. Whole bone marrow cells was collected from BALB/c mice 4 days after 5-FU treatment and transduced with P140KMGMT-luc lentiviruses at MOI of 1.4. Transduced bone marrow cells were transplanted into lethally irradiated or non-myeloablated syngeneic recipient mice at different cell numbers. Initial bioluminescent signal emerged 6–8 days after transplantation in both lethally irradiated and non-myeloablated recipients. The onset of bioluminescent foci after transplantation occurred in a cell dose dependent manner. The initial signal emitted predominantly from bone marrow, especially femurs, humeri and vertebrae during the early stage of clonal expansion. Intense signal appeared in spleen at days 12–14 and became weaker or even disappeared by days 20–28. Clonal expansion and engraftment greatly increased after a single course of BG+TMZ treatment and initiated strong hematopoiesis in non-myeloablated recipients. Total body bioluminescence intensity of drug treated mice increased 24 fold and 7 fold compared to non-treated mice in both non-myeloablated and lethally irradiated recipients, respectively. A transient phase suggesting migration through the lymphatic system and in the spleen occurred in most mice and was exacerbated by drug selection, but this was less clear in lethally irradiated mice, where engraftment was more confined to the marrow spaces. Bioluminescence in vivo imaging reveals active migration between the bone marrow and the spleen during hematopoiesis. Drug selection has a significant impact on the patterns of engraftment and clonal expansion of HSC and progenitor cells after transplantation.

Hmgb3 Deficiency Inhibits Down-Regulation of c-kit in Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 372-372
Author(s):  
Michael J. Nemeth ◽  
Stacie M. Anderson ◽  
Lisa J. Garrett-Beal ◽  
David M. Bodine
Keyword(s):  
Bone Marrow ◽  
Hind Limb ◽  
Bone Marrow Cells ◽  
Es Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Bone Marrow Cellularity ◽  
Marrow Cellularity ◽  
Marrow Cells

Abstract Hmgb3 is an X-linked member of a family of sequence-independent chromatin-binding proteins that is expressed in HSC-enriched lin−, c-kitHI, Sca-1HI, IL-7Rα− (KSIL) cells and Ter119+ erythroid cells. To define Hmgb3 function, we generated hemizygous mice (Hmgb3−/Y) using 129/SvJ ES cells. Hmgb3−/Y mice contain normal numbers of KSIL cells that are capable of normal repopulation and self-renewal. However, these mice have 1.6-fold fewer common lymphoid progenitors (CLP) and 3-fold fewer common myeloid progenitors (CMP) (p < 0.05). We hypothesized that the role of Hmgb3 in early hematopoiesis involves c-kit regulation. We observed that the level of c-kit mRNA in Hmgb3−/Y HSCs increased 30% compared to wild-type (WT) (p = 0.05). We used 5-fluorouracil (5-FU), which has been shown to down-regulate c-kit on HSCs, to characterize the interaction between Hmgb3 and c-kit. We monitored Hmgb3 expression in KSIL and lin−, Sca-1+, c-kit− cells before and after 5-FU treatment (150 mg/kg) using phenotypically normal transgenic mice containing an IRES-GFP cassette knocked into the 3′ UTR of Hmgb3. Prior to 5-FU treatment, 27% of KSIL cells were GFP+ (these cells were absent 4 days post-injection {p.i.}). In contrast, 1.8% of lin−, c-kit−, Sca-1+ cells were GFP+ before 5-FU treatment whereas 26% of lin−, c-kit−, Sca-1+ cells were GFP+ 4 days p.i. The increased proportion of GFP+ lin-, c-kit−, Sca-1+ cells after 5-FU treatment is consistent with previous findings that repopulating activity resides within the c-kit−/LO population in 5-FU treated bone marrow and our finding that Hmgb3 serves as a marker for long-term repopulating activity. To determine the time course of c-kit regulation, we compared bone marrow from 5-FU injected Hmgb3−/Y and WT mice for analysis at 2, 4, and 6 days p.i. Two days p.i., both WT and Hmgb3−/Y mice contained similar numbers of bone marrow cells (7 x 106 cells/hind limb) and the KSIL population was absent. By four days p.i., the bone marrow cellularity of WT mice declined to 5.5 ± 0.9 x 106 cells/hind limb and KSIL cells were still absent. However, in Hmgb3−/Y mice 4 days p.i., bone marrow cellularity stabilized at 7.9 ± 0.8 x 106 cells/hind limb, an increase of 43% compared to WT (p < 0.01), along with the re-emergence of the KSIL population. To determine whether the Hmgb3−/Y lin−, c-kit−, Sca-1+ population contains repopulating HSCs after 4 days of 5-FU treatment similar to WT mice, we performed repopulation assays using KSIL and lin−, c-kit−, Sca-1+ cells sorted from 4 day p.i. 5-FU treated Hmgb3−/Y mice. Recipients received either 2 x 104 KSIL or 2 x 105 lin−, c-kit−, Sca-1+ cells (Ly 5.2) from 5-FU treated Hmgb3−/Y mice along with a radioprotective dose of 3 x 105 congenic (Ly 5.1) bone marrow cells. FACS analysis performed on control recipients transplanted with congenic marrow exhibited < 1% Ly 5.2 cells in the bone marrow 16 weeks after transplant. Pre-5-FU treatment, 88% of bone marrow cells were donor derived in recipients of Hmgb3−/Y KSIL cells. There was no detectable engraftment of Hmgb3−Y lin−, c-kit−, Sca-1+ cells. In contrast to WT mice, both KSIL and lin−, c-kit−, Sca-1+ cells from 5-FU treated Hmgb3−/Y mice were capable of long-term repopulation (62–82% donor derived cells). We conclude that Hmgb3 deficiency facilitates the reemergence of c-kitHI HSCs following 5-FU treatment. Mechanisms involving either enhanced HSC self-renewal or delayed differentiation into CLPs and CMPs are both consistent with our results.

Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3620-3627 ◽  
Author(s):  
Graça Almeida-Porada ◽  
Christopher D. Porada ◽  
Nam Tran ◽  
Esmail D. Zanjani
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
In Utero ◽  
Human Cells ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Hsc Transplantation

Both in utero and postnatal hematopoietic stem cell (HSC) transplantation would benefit from the development of approaches that produce increased levels of engraftment or a reduction in the period of time required for reconstitution. We used the in utero model of human–sheep HSC transplantation to investigate ways of improving engraftment and differentiation of donor cells after transplantation. We hypothesized that providing a more suitable microenvironment in the form of human stromal cell progenitors simultaneously with the transplanted human HSC would result in higher rates of engraftment or differentiation of the human cells in this xenogeneic model. The results presented here demonstrate that the cotransplantation of both autologous and allogeneic human bone marrow-derived stromal cell progenitors resulted in an enhancement of long-term engraftment of human cells in the bone marrow of the chimeric animals and in earlier and higher levels of donor cells in circulation both during gestation and after birth. By using marked stromal cells, we have also demonstrated that injected stromal cells alone engraft and remain functional within the sheep hematopoietic microenvironment. Application of this method to clinical HSC transplantation could potentially lead to increased levels of long-term engraftment, a reduction in the time for hematopoietic reconstitution, and a means of delivery of foreign genes to the hematopoietic system.

scholarly journals HIF-1α is a negative regulator of plasmacytoid DC development in vitro and in vivo

Blood ◽  
2012 ◽  
Vol 120 (15) ◽  
pp. 3001-3006 ◽  
Author(s):  
Andreas Weigert ◽  
Benjamin Weichand ◽  
Divya Sekar ◽  
Weixiao Sha ◽  
Christina Hahn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Function ◽  
Bone Marrow Cells ◽  
Negative Regulator ◽  
Low Oxygen ◽  
Hematopoietic Stem ◽  
Lineage Differentiation ◽  
Marrow Cells

Abstract Hypoxia-inducible factors (HIFs) regulate hematopoiesis in the embryo and maintain hematopoietic stem cell function in the adult. How hypoxia and HIFs contribute to hematopoietic lineage differentiation in the adult is ill defined. Here we provide evidence that HIF-1 limits differentiation of precursors into plasmacytoid dendritic cells (pDCs). Low oxygen up-regulated inhibitor of DNA binding 2 (ID2) and suppressed Flt3-L–induced differentiation of bone marrow cells to pDCs in wild-type but not HIF-1αfl/fl LysM-Cre bone marrow cells. Moreover, pDC differentiated normally in hypoxic ID2−/− bone marrow cultures. Finally, we observed elevated pDC frequencies in bone marrow, blood, and spleen of HIF-1αfl/fl LysM-Cre and ID2−/−, but not HIF-2αfl/fl LysM-Cre mice. Our data indicate that the low oxygen content in the bone marrow might limit pDC development. This might be an environmental mechanism to restrict the numbers of these potentially autoreactive cells.

scholarly journals Divisional Memory Drives Hematopoietic Stem Cell Functional Diversity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 20-20
Author(s):  
James Bartram ◽  
Baobao (Annie) Song ◽  
Juying Xu ◽  
Nathan Salomonis ◽  
H. Leighton Grimes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Functional Decline ◽  
Functional Heterogeneity ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Marrow Cells

Abstract Hematopoietic stem cells are endowed with high regenerative potential but their actual self-renewal capacity is limited. Studies using the H2B-retention labeling system show HSC functional decline at each round of division (Qiu, Stem Cell Reports 2014). We have shown that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here we examined the link between mitochondrial metabolism, in vivo division at steady state, and HSC functions using the GFP label-Histone 2B (GFP-H2B) mouse model driven by a doxycycline-inducible promoter. Five months after doxycycline removal, mitochondrial membrane potential (MMP) was examined using TMRE in HSC with varying GFP intensity. HSC were separated into an H2B-labeled retention population and an H2B-labeled population. Interestingly, within the H2B-labeled retention population, HSC could be further subdivided into GFP high, medium, and low. MMP increased in a stepwise fashion with GFP dilution in HSC. We noted the presence of 2 TMRE peaks within each GFP high and medium populations leading to 5 populations: GFP-high;MMP-low (G1), GFP-high;MMP-high (G2), GFP-medium;MMP-low (G3), GFP-medium;MMP-high (G4), GFP-low;MMP-high (G5). We examined the repopulation activity of each population in a serial competitive transplant assay. G1 and G2 maintained higher peripheral blood chimerism up to 24 weeks post-transplant than G3 and G4. G5 did not engraft at all. However, only G1 reconstituted high frequency of HSC in primary recipients. In secondary recipients, G1, G2, G3 but not G4 gave rise to positive engraftment. Interestingly, G1 and G2 grafts showed myeloid/lymphoid balanced engraftment whereas the G3 graft was myeloid-bias, suggesting that myeloid skewing can be acquired upon HSC division. We further examined lineage fate maps of bone marrow cells derived from G1 or G3 population in vivo, using single cell RNA sequencing, 10X genomics. Surprisingly, G3-derived bone marrow cells displayed a distinct myeloid cell trajectory from G1-derived bone marrow cells, in which G3 gave rise to increased immature neutrophils but fewer myeloid precursors. Remarkably, each lineage population derived from G3 donor cells had different gene expression signatures than those derived from G1 donor cells. Therefore, HSC that have divided in vivo in the same bone marrow microenvironment are intrinsically and molecularly different such that not only do they exhibit lineage potential differences but they also produce progeny that are transcriptionally different. These findings imply that cellular division rewires HSC and that this rewiring is passed down to their fully differentiated progeny. When G1 and G3 single HSC were cultured in-vitro, G1 had a slower entry into cell-cycle which has been associated with increased stemness. Additionally, when single HSC from G1 and G3 were assessed for their multipotency in a lineage differentiation assay, G1 HSC had a higher propensity to produce all four myeloid lineages (megakaryocytes, neutrophils, macrophages, and erythroid), further supporting increased stemness in G1 compared to G3 HSC. Finally, HSC from G1, G2, G3 and G4 populations carried mitochondria that were morphologically different, and express distinct levels of Sca-1, CD34 and EPCR, with Sca-1 high, CD34-, EPCR+ cells more enriched in G1. In summary, this study suggests that HSC transition into distinct metabolic and functional states with division history that may contribute to HSC diversity and functional heterogeneity. It also suggests the existence of a cell-autonomous mechanism that confers HSC divisional memory to actively drive HSC functional heterogeneity and aging. Disclosures No relevant conflicts of interest to declare.

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