Long term follow-up after haematopoietic stem cell transplantation for mucopolysaccharidosis type I-H: a retrospective study of 51 patients.

Mucopolysaccharidosis type I-H (MPS I-H) is a rare lysosomal storage disorder caused by α-L-Iduronidase deficiency. Early haematopoietic stem cell transplantation (HSCT) is the sole available therapeutic option to preserve neurocognitive functions. We report long-term follow-up (median 9 years, interquartile range 8-16.5) for 51 MPS I-H patients who underwent HSCT between 1986 and 2018 in France. 4 patients died from complications of HSCT and one from disease progression. Complete chimerism and normal α-L-Iduronidase activity were obtained in 84% and 71% of patients respectively. No difference of outcomes was observed between bone marrow and cord blood stem cell sources. All patients acquired independent walking and 91% and 78% acquired intelligible language or reading and writing. Intelligence Quotient evaluation (n = 23) showed that 69% had IQ ≥ 70 at last follow-up. 58% of patients had normal or remedial schooling and 62% of the 13 adults had good socio-professional insertion. Skeletal dysplasia as well as vision and hearing impairments progressed despite HSCT, with significant disability. These results provide a long-term assessment of HSCT efficacy in MPS I-H and could be useful in the evaluation of novel promising treatments such as gene therapy.

Initial presentation, management and follow-up data of 33 treated patients with hereditary tyrosinemia type 1 in the absence of newborn screening.

Hereditary tyrosinemia type 1 (HT1) is a rare autosomal recessive disorder of phenylalanine and tyrosine catabolism due to a deficiency of fumarylacetoacetate hydrolase. HT1 has a large clinical spectrum with acute forms presenting before six months of age, subacute forms with initial symptoms occurring between age 6 and 12 months, and chronic forms after 12 months of age. Without treatment, HT1 results in the accumulation of toxic metabolites leading to liver disease, proximal tubular dysfunction, and porphyria-like neurological crises. Since the early nineties, the outcome of HT1 has dramatically changed due to its treatment with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC, nitisinone). In some countries, HT1 is included in the newborn screening program based on the analysis of succinylacetone concentration on dried blood spots. In the present study, we report clinical and laboratory parameters data on 33 HT1 patients focusing on clinical presentation and therapeutic management at the time of diagnosis. Eighteen patients were diagnosed with the acute form (median age at presentation 2.5 months), 6 with the subacute form (median age at presentation 10 months), and 5 with the chronic form of HT1 (median age at presentation 15 months). Four patients were diagnosed pre-symptomatically in the setting of a family history of HT1. Among the 29 symptomatic patients, hepatomegaly was found in 83% of patients and prolonged coagulation times due to hepatocellular insufficiency was observed in 93% of patients. HT1 diagnosis was confirmed by increased urine succinylacetone in all patients. All patients but 2 were treated with nitisinone immediately at diagnosis. During follow-up, 2 patients received liver transplant for high grade dysplasia or hepatocellular carcinoma, 10 patients exhibited some form of neurocognitive impairments. Our data confirm that HT1 is a severe treatable liver disease that should be detected at the earliest, ideally by newborn screening and appropriately treated.

Oncostatin M regulates hematopoietic stem cell (HSC) niches in the bone marrow to restrict HSC mobilization.

We show that pro-inflammatory oncostatin M (OSM) is an important regulator of hematopoietic stem cell (HSC) niches in the bone marrow (BM). Treatment of healthy humans and mice with granulocyte colony-stimulating factor (G-CSF) dramatically increases OSM release in blood and BM. Using mice null for the OSM receptor (OSMR) gene, we demonstrate that OSM provides a negative feed-back acting as a brake on HSPC mobilization in response to clinically relevant mobilizing molecules G-CSF and CXCR4 antagonist. Likewise, injection of a recombinant OSM molecular trap made of OSMR complex extracellular domains enhances HSC mobilization in poor mobilizing C57BL/6 and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Mechanistically, OSM attenuates HSC chemotactic response to CXCL12 and increases HSC homing to the BM signaling indirectly via BM endothelial and mesenchymal cells which are the only cells expressing OSMR in the BM. OSM up-regulates E-selectin expression on BM endothelial cells indirectly increasing HSC proliferation. RNA sequencing of HSCs from Osmr-/- and wild-type mice suggest that HSCs have altered cytoskeleton reorganization, energy usage and cycling in the absence of OSM signaling in niches. Therefore OSM is an important regulator of HSC niche function restraining HSC mobilization and anti-OSM therapy combined with current mobilizing regimens may improve HSPC mobilization for transplantation.

Prostacyclin is an endosteal bone marrow niche component and its clinical analog iloprost protects hematopoietic stem cell potential during stress.

Hematopoietic stem cells (HSCs) with superior reconstitution potential are reported to be enriched in the endosteal compared to central bone marrow (BM) region. To investigate whether specific factors at the endosteum may contribute to HSC potency, we screened for candidate HSC niche factors enriched in the endosteal compared to central BM regions. Together with key known HSC supporting factors Kitl and Cxcl12, we report that prostacyclin/prostaglandin I2 (PGI2 ) synthase (Ptgis) was one of the most highly enriched mRNAs (>10-fold) in endosteal compared to central BM. As PGI2 signals through receptors distinct from prostaglandin E2 (PGE2 ), we investigated functional roles for PGI2 at the endosteal niche using therapeutic PGI2 analogs, iloprost, and cicaprost. We found PGI2 analogs strongly reduced HSC differentiation in vitro. Ex vivo iloprost pulse treatment also significantly boosted long-term competitive repopulation (LT-CR) potential of HSCs upon transplantation. This was associated with increased tyrosine-phosphorylation of transducer and activator of transcription-3 (STAT3) signaling in HSCs but not altered cell cycling. In vivo, iloprost administration protected BM HSC potential from radiation or granulocyte colony-stimulating factor-induced exhaustion, and restored HSC homing potential with increased Kitl and Cxcl12 transcription in the BM. In conclusion, we propose that PGI2 is a novel HSC regulator enriched in the endosteum that promotes HSC regenerative potential following stress.

Bacterial Lipopolysaccharides Suppress Erythroblastic Islands and Erythropoiesis in the Bone Marrow in an Extrinsic and G- CSF-, IL-1-, and TNF-Independent Manner.

Anemia of inflammation (AI) is the second most prevalent anemia after iron deficiency anemia and results in persistent low blood erythrocytes and hemoglobin, fatigue, weakness, and early death. Anemia of inflammation is common in people with chronic inflammation, chronic infections, or sepsis. Although several studies have reported the effect of inflammation on stress erythropoiesis and iron homeostasis, the mechanisms by which inflammation suppresses erythropoiesis in the bone marrow (BM), where differentiation and maturation of erythroid cells from hematopoietic stem cells (HSCs) occurs, have not been extensively studied. Here we show that in a mouse model of acute sepsis, bacterial lipopolysaccharides (LPS) suppress medullary erythroblastic islands (EBIs) and erythropoiesis in a TLR-4- and MyD88-dependent manner with concomitant mobilization of HSCs. LPS suppressive effect on erythropoiesis is indirect as erythroid progenitors and erythroblasts do not express TLR-4 whereas EBI macrophages do. Using cytokine receptor gene knock-out mice LPS-induced mobilization of HSCs is G-CSF-dependent whereas LPS-induced suppression of medullary erythropoiesis does not require G- CSF-, IL- 1-, or TNF-mediated signaling. Therefore suppression of medullary erythropoiesis and mobilization of HSCs in response to LPS are mechanistically distinct. Our findings also suggest that EBI macrophages in the BM may sense innate immune stimuli in response to acute inflammation or infections to rapidly convert to a pro-inflammatory function at the expense of their erythropoietic function.

Acute Myeloid Leukemia Chemo-Resistance Is Mediated by E-selectin Receptor CD162 in Bone Marrow Niches.

The interactions of leukemia cells with the bone marrow (BM) microenvironment is critical for disease progression and resistance to treatment. We have recently found that the vascular adhesion molecule E-(endothelial)-selectin is a key niche component that directly mediates acute myeloid leukemia (AML) chemo-resistance, revealing E-selectin as a promising therapeutic target. To understand how E-selectin promotes AML survival, we investigated the potential receptors on AML cells involved in E-selectin-mediated chemo-resistance. Using CRISPR-Cas9 gene editing to selectively suppress canonical E-selectin receptors CD44 or P-selectin glycoprotein ligand-1 (PSGL-1/CD162) from human AML cell line KG1a, we show that CD162, but not CD44, is necessary for E-selectin-mediated chemo-resistance in vitro. Using preclinical models of murine AML, we then demonstrate that absence of CD162 on AML cell surface leads to a significant delay in the onset of leukemia and a significant increase in sensitivity to chemotherapy in vivo associated with a more rapid in vivo proliferation compared to wild-type AML and a lower BM retention. Together, these data reveal for the first time that CD162 is a key AML cell surface receptor involved in AML progression, BM retention and chemo-resistance. These findings highlight specific blockade of AML cell surface CD162 as a potential novel niche-based strategy to improve the efficacy of AML therapy.

Endothelial E-selectin inhibition improves acute myeloid leukaemia therapy by disrupting vascular niche-mediated chemoresistance.

The endothelial cell adhesion molecule E-selectin is a key component of the bone marrow hematopoietic stem cell (HSC) vascular niche regulating balance between HSC self-renewal and commitment. We now report in contrast, E-selectin directly triggers signaling pathways that promote malignant cell survival and regeneration. Using acute myeloid leukemia (AML) mouse models, we show AML blasts release inflammatory mediators that upregulate endothelial niche E-selectin expression. Alterations in cell-surface glycosylation associated with oncogenesis enhances AML blast binding to E-selectin and enable promotion of pro-survival signaling through AKT/NF-κB pathways. In vivo AML blasts with highest E-selectin binding potential are 12-fold more likely to survive chemotherapy and main contributors to disease relapse. Absence (in Sele-/- hosts) or therapeutic blockade of E-selectin using small molecule mimetic GMI-1271/Uproleselan effectively inhibits this niche-mediated pro-survival signaling, dampens AML blast regeneration, and strongly synergizes with chemotherapy, doubling the duration of mouse survival over chemotherapy alone, whilst protecting endogenous HSC.

Mobilization of hematopoietic stem cells with highest self-renewal by G-CSF precedes clonogenic cell mobilization peak.

Harvest of granulocyte colony-stimulating factor (G-CSF)-mobilized hematopoietic stem cells (HSCs) begins at day 5 of G-CSF administration, when most donors have achieved maximal mobilization. This is based on surrogate markers for HSC mobilization, such as CD34(+) cells and colony-forming activity in blood. However, CD34(+) cells or colony-forming units in culture (CFU-C) are heterogeneous cell populations with hugely divergent long-term repopulation potential on transplantation. HSC behavior is influenced by the vascular bed in the vicinity of which they reside. We hypothesized that G-CSF may mobilize sequentially cells proximal and more distal to bone marrow venous sinuses where HSCs enter the blood. We addressed this question with functional serial transplantation assays using blood and bone marrow after specific time points of G-CSF treatment in mice. We found that in mice, blood collected after only 48 hours of G-CSF administration was as enriched in serially reconstituting HSCs as blood collected at 5 days of G-CSF treatment. Similarly, mobilized Lin(-)CD34(+) cells were relatively enriched in more primitive Lin(-)CD34(+)CD38(-) cells at day 2 of G-CSF treatment compared with later points in half of human donors tested (n = 6). This suggests that in both humans and mice, hematopoietic progenitor and stem cells do not mobilize uniformly according to their maturation stage, with most potent HSCs mobilizing as early as day 2 of G-CSF.

Fms-like tyrosine kinase 3 (Flt3) ligand depletes erythroid island macrophages and blocks medullar erythropoiesis in the mouse.

The cytokines granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (Flt3-L) mobilize hematopoietic stem and progenitor cells into the peripheral blood of primates, humans, and mice. We recently reported that G-CSF administration causes a transient blockade of medullar erythropoiesis by suppressing erythroblastic island (EI) macrophages in the bone marrow. In the study described here, we investigated the effect of mobilizing doses of Flt3-L on erythropoiesis in mice in vivo. Similar to G-CSF, Flt3-L caused whitening of the bone marrow with significant reduction in the numbers of EI macrophages and erythroblasts. This was compensated by an increase in the numbers of EI macrophages and erythroblasts in the spleen. However, unlike G-CSF, Flt3-L had an indirect effect on EI macrophages, as it was not detected at the surface of EI macrophages or erythroid progenitors.

Tissue engineered humanized bone supports human hematopoiesis in vivo.

Advances in tissue-engineering have resulted in a versatile tool-box to specifically design a tailored microenvironment for hematopoietic stem cells (HSCs) in order to study diseases that develop within this setting. However, most current in vivo models fail to recapitulate the biological processes seen in humans. Here we describe a highly reproducible method to engineer humanized bone constructs that are able to recapitulate the morphological features and biological functions of the HSC niches. Ectopic implantation of biodegradable composite scaffolds cultured for 4 weeks with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone organ including a large number of human mesenchymal cells which were shown to be metabolically active and capable of establishing a humanized microenvironment supportive of the homing and maintenance of human HSCs. A syngeneic mouse-to-mouse transplantation assay was used to prove the functionality of the tissue-engineered ossicles. We predict that the ability to tissue engineer a morphologically intact and functional large-volume bone organ with a humanized bone marrow compartment will help to further elucidate physiological or pathological interactions between human HSCs and their native niches.

Neurological heterotopic ossification following spinal cord injury is triggered by macrophage-mediated inflammation in muscle.

Neurological heterotopic ossification (NHO) is the abnormal formation of bone in soft tissues as a consequence of spinal cord or traumatic brain injury. NHO causes pain, ankyloses, vascular and nerve compression and delays rehabilitation in this high-morbidity patient group. The pathological mechanisms leading to NHO remain unknown and consequently there are no therapeutic options to prevent or reduce NHO. Genetically modified mouse models of rare genetic forms of heterotopic ossification (HO) exist, but their relevance to NHO is questionable. Consequently, we developed the first model of spinal cord injury (SCI)-induced NHO in genetically unmodified mice. Formation of NHO, measured by micro-computed tomography, required the combination of both SCI and localized muscular inflammation. Our NHO model faithfully reproduced many clinical features of NHO in SCI patients and both human and mouse NHO tissues contained macrophages. Muscle-derived mesenchymal progenitors underwent osteoblast differentiation in vitro in response to serum from NHO mice without additional exogenous osteogenic stimuli. Substance P was identified as a candidate NHO systemic neuropeptide, as it was significantly elevated in the serum of NHO patients. However, antagonism of substance P receptor in our NHO model only modestly reduced the volume of NHO. In contrast, ablation of phagocytic macrophages with clodronate-loaded liposomes reduced the size of NHO by 90%, supporting the conclusion that NHO is highly dependent on inflammation and phagocytic macrophages in soft tissues. Overall, we have developed the first clinically relevant model of NHO and demonstrated that a combined insult of neurological injury and soft tissue inflammation drives NHO pathophysiology.

Mobilization with granulocyte colony-stimulating factor blocks medullar erythropoiesis by depleting F4/80(+)VCAM1(+)CD169(+)ER-HR3(+)Ly6G(+) erythroid island macrophages in the mouse.

Similarly to other tissues, the bone marrow contains subsets of resident tissue macrophages, which are essential to maintain bone formation, functional hematopoietic stem cell (HSC) niches, and erythropoiesis. Pharmacologic doses of granulocyte colony-stimulating factor (G-CSF) mobilize HSC in part by interfering with the HSC niche-supportive function of BM resident macrophages. Because bone marrow macrophages are key to both maintenance of HSC within their niche and erythropoiesis, we investigated the effect of mobilizing doses of G-CSF on erythropoiesis in mice. We now report that G-CSF blocks medullar erythropoiesis by depleting the erythroid island macrophages we identified as co-expressing F4/80, vascular cell adhesion molecule-1, CD169, Ly-6G, and the ER-HR3 erythroid island macrophage antigen. Both broad macrophage depletion, achieved by injecting clodronate-loaded liposomes, and selective depletion of CD169(+) macrophages, also concomitantly depleted F4/80(+)VCAM-1(+)CD169(+)ER-HR3(+)Ly-6G(+) erythroid island macrophages and blocked erythropoiesis. This more precise phenotypic definition of erythroid island macrophages will enable studies on their biology and function in normal settings and on diseases associated with anemia. Finally, this study further illustrates that macrophages are a potent relay of innate immunity and inflammation on bone, hematopoietic, and erythropoietic maintenance. Agents that affect these macrophages, such as G-CSF, are likely to affect these three processes concomitantly.

Interaction of c-Myb with p300 is required for the induction of acute myeloid leukemia (AML) by human AML oncogenes.

The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, suggesting that MYB may be a therapeutic target in these diseases. However, realization of this potential requires a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis, and an approach for developing an effective therapeutic. We previously showed that the interaction of c-Myb with the coactivator CBP/p300 is essential for its transforming activity. Here, by using cells from Booreana mice which carry a mutant allele of c-Myb, we show that this interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type mice, Booreana cells transduced with AML1-ETO9a or MLL-AF9 retroviruses fail to generate leukemia upon transplantation into irradiated recipients. Finally, we have begun to explore the molecular mechanisms underlying these observations by gene expression profiling. This identified several genes previously implicated in myeloid leukemogenesis and HSC function as being regulated in a c-Myb-p300-dependent manner. These data highlight the importance of the c-Myb-p300 interaction in myeloid leukemogenesis and suggest disruption of this interaction as a potential therapeutic strategy for acute myeloid leukemia.

Pharmacologic stabilization of HIF-1α increases hematopoietic stem cell quiescence in vivo and accelerates blood recovery after severe irradiation.

UNLABELLED: Quiescent hematopoietic stem cells (HSCs) preferentially reside in poorly perfused niches that may be relatively hypoxic. Most of the cellular effects of hypoxia are mediated by O2-labile hypoxia-inducible transcription factors (HIFs). To investigate the effects of hypoxia on HSCs, we blocked O2-dependent HIF-1α degradation in vivo in mice by injecting 2 structurally unrelated prolyl hydroxylase domain (PHD) enzyme inhibitors: dimethyloxalyl glycine and FG-4497. Injection of either of these 2 PHD inhibitors stabilized HIF-1α protein expression in the BM. In vivo stabilization of HIF-1a with these PHD inhibitors increased the proportion of phenotypic HSCs and immature hematopoietic progenitor cells in phase G0 of the cell cycle and decreased their proliferation as measured by 5-bromo-2'-deoxyuridine incorporation. This effect was independent of erythropoietin, the expression of which was increased in response to PHD inhibitors. Finally, pretreatment of mice with a HIF-1α stabilizer before severe, sublethal 9.0-Gy irradiation improved blood recovery and enhanced 89-fold HSC survival in the BM of irradiated mice as measured in long-term competitive repopulation assays. The results of the present study demonstrate that the levels of HIF-1α protein can be manipulated pharmacologically in vivo to increase HSC quiescence and recovery from irradiation. KEY POINTS: HIF-1α protein stabilization increases HSC quiescence in vivo. HIF-1α protein stabilization increases HSC resistance to irradiation and accelerates recovery.

B-lymphopoiesis is stopped by mobilizing doses of G-CSF and is rescued by overexpression of the anti-apoptotic protein Bcl2.

Osteoblasts are necessary to B lymphopoiesis and mobilizing doses of G-CSF or cyclophosphamide inhibit osteoblasts, whereas AMD3100/Plerixafor does not. However, the effect of these mobilizing agents on B lymphopoiesis has not been reported. Mice (wild-type, knocked-out for TNF-α and TRAIL, or over-expressing Bcl-2) were mobilized with G-CSF, cyclophosphamide, or AMD3100. Bone marrow, blood, spleen and lymph node content in B cells was measured. G-CSF stopped medullar B lymphopoiesis with concomitant loss of B-cell colony-forming units, pre-pro-B, pro-B, pre-B and mature B cells and increased B-cell apoptosis by an indirect mechanism. Overexpression of the anti-apoptotic protein Bcl2 in transgenic mice rescued B-cell colony forming units and pre-pro-B cells in the marrow, and prevented loss of all B cells in marrow, blood and spleen. Blockade of endogenous soluble TNF-α with Etanercept, or combined deletion of the TNF-α and TRAIL genes did not prevent B lymphopoiesis arrest in response to G-CSF. Unlike G-CSF, treatments with cyclophosphamide or AMD3100 did not suppress B lymphopoiesis but caused instead robust B-cell mobilization. G-CSF, cyclophosphamide and AMD3100 have distinct effects on B lymphopoiesis and B-cell mobilization with: 1) G-CSF inhibiting medullar B lymphopoiesis without mobilizing B cells in a mechanism distinct from the TNF-α-mediated loss of B lymphopoiesis observed during inflammation or viral infections; 2) CYP mobilizing B cells but blocking their maturation; and 3) AMD3100 mobilizing B cells without affecting B lymphopoiesis. These results suggest that blood mobilized with these three agents may have distinct immune properties.

Engraftment Outcomes after HPC Co-Culture with Mesenchymal Stromal Cells and Osteoblasts.

Haematopoietic stem cell (HSC) transplantation is an established cell-based therapy for a number of haematological diseases. To enhance this therapy, there is considerable interest in expanding HSCs in artificial niches prior to transplantation. This study compared murine HSC expansion supported through co-culture on monolayers of either undifferentiated mesenchymal stromal cells (MSCs) or osteoblasts. Sorted Lineage(-) Sca-1(+) c-kit(+) (LSK) haematopoietic stem/progenitor cells (HPC) demonstrated proliferative capacity on both stromal monolayers with the greatest expansion of LSK shown in cultures supported by osteoblast monolayers. After transplantation, both types of bulk-expanded cultures were capable of engrafting and repopulating lethally irradiated primary and secondary murine recipients. LSKs co-cultured on MSCs showed comparable, but not superior, reconstitution ability to that of freshly isolated LSKs. Surprisingly, however, osteoblast co-cultured LSKs showed significantly poorer haematopoietic reconstitution compared to LSKs co-cultured on MSCs, likely due to a delay in short-term reconstitution. We demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSCs without a need for additional haematopoietic growth factors. We also demonstrated that despite apparently superior in vitro performance, co-injection of bulk cultures of osteoblasts and LSKs in vivo was detrimental to recipient survival and should be avoided in translation to clinical practice.

Vascular niche E-selectin regulates hematopoietic stem cell dormancy, self renewal and chemoresistance.

The microenvironment, or niche, surrounding a stem cell largely governs its cellular fate. Two anatomical niches for hematopoietic stem cells (HSCs) have been reported in the bone marrow, but a distinct function for each of these niches remains unclear. Here we report a new role for the adhesion molecule E-selectin expressed exclusively by bone marrow endothelial cells in the vascular HSC niche. HSC quiescence was enhanced and self-renewal potential was increased in E-selectin knockout (Sele(-/-)) mice or after administration of an E-selectin antagonist, demonstrating that E-selectin promotes HSC proliferation and is a crucial component of the vascular niche. These effects are not mediated by canonical E-selectin ligands. Deletion or blockade of E-selectin enhances HSC survival threefold to sixfold after treatment of mice with chemotherapeutic agents or irradiation and accelerates blood neutrophil recovery. As bone marrow suppression is a severe side effect of high-dose chemotherapy, transient blockade of E-selectin is potentially a promising treatment for the protection of HSCs during chemotherapy or irradiation.

Mobilization of hematopoietic stem cells by depleting bone marrow macrophages.

An important factor contributing to hematopoietic stem cell (HSC) mobilization is the ability of mobilizing cytokines and chemotherapy to disturb the cellular components of HSC niches, particularly osteoblasts and their progenitors, and to inhibit the production of HSC supportive cytokines and chemokines. Although the mechanisms by which niche cells are inhibited by mobilizing treatments is still incompletely understood, it has recently emerged that bone marrow macrophages play a critical role in maintaining osteoblasts, bone formation, and the expression of CXCL12, KIT ligand, and angiopoietin-1 necessary to HSC maintenance. In this chapter, we describe how to mobilize HSC into the blood in mice by depleting macrophages with clodronate-loaded liposomes and compare this mode of mobilization to mobilization induced by granulocyte colony-stimulating factor and cyclophosphamide. Detailed methods to analyze mobilization of phenotypic and functional reconstituting HSC are described with examples.

Flow cytometry analysis of cell cycling and proliferation in mouse hematopoietic stem and progenitor cells.

The hematopoietic system is highly proliferative in the bone marrow (BM) due to the short half-life of granulocytes and platelets in the blood. Analysis of cell cycling and cell proliferation in vivo in specific populations of the mouse BM has highlighted some key properties of adult hematopoietic stem cells (HSCs). For instance, despite their enormous proliferation and repopulation potential, most true HSC are deeply quiescent in G(0) phase of the cell cycle and divide very infrequently, while less potent lineage-restricted progenitors divide rapidly to replace the daily consumption of blood leukocytes, erythrocytes, and platelets. In response to stress, e.g., following ablative chemotherapy or irradiation, HSC must enter the cell cycle to rapidly repopulate the BM with progenitors. Due to their extreme rarity in the BM, at least five color flow cytometry for cell surface antigens has to be combined with staining for DNA content and nuclear markers of proliferation to analyze cell cycle and proliferation of HSC in vivo. In this chapter, we describe two methods to stain mouse HSC to (1) distinguish all phases of the cell cycle (G(0), G(1), S, and G(2)/M) and (2) analyze the divisional history of HSC in vivo by incorporation of the thymidine analog 5-bromo-2-deoxyuridine.

Flow cytometry measurement of bone marrow perfusion in the mouse and sorting of progenitors and stems cells according to position relative to blood flow in vivo.

Identification of the precise location, where hematopoietic stem cells (HSCs) reside in the bone marrow, has made a great leap forward with the advance of live time-lapse video 2-photon fluorescent microscopy. These studies have shown that HSCs preferentially resides in the endosteal region of the BM, at an average of two cell diameters from osteoblasts covering endosteal bone surfaces. However, this equipment is very sophisticated and only a very few laboratories can perform these studies. To investigate functional attributes of these niches, we have developed a flow cytometry technique in which mice are perfused with the cell-permeable fluorescent dye Hoechst33342 in vivo before bone marrow cells are collected and antibody stained. This method enables to position phenotypic HSC, multipotent and myeloid progenitors, as well as BM nonhematopoietic stromal cells relative to blood flow in vivo. This technique enables prospective isolation of HSCs based on the in vivo perfusion of the niches in which they reside.