Physioxia-induced downregulation of Tet2 in hematopoietic stem cells contributes to enhanced self-renewal.

Hematopoietic stem cells (HSCs) manifest impaired recovery and self-renewal with a concomitant increase in differentiation when exposed to ambient air as opposed to physioxia. Mechanism(s) behind this distinction are poorly understood but have the potential to improve stem cell transplantation. Single-cell RNA sequencing of HSCs in physioxia revealed upregulation of HSC self-renewal genes and downregulation of genes involved in inflammatory pathways and HSC differentiation. HSCs under physioxia also exhibited downregulation of the epigenetic modifier Tet2. Tet2 is α-ketoglutarate, iron- and oxygen-dependent dioxygenase that converts 5-methylcytosine to 5-hydroxymethylcytosine, thereby promoting active transcription. We evaluated whether loss of Tet2 affects the number and function of HSCs and hematopoietic progenitor cells (HPCs) under physioxia and ambient air. In contrast to wild-type HSCs (WT HSCs), a complete nonresponsiveness of Tet2-/- HSCs and HPCs to changes in oxygen tension was observed. Unlike WT HSCs, Tet2-/- HSCs and HPCs exhibited similar numbers and function in either physioxia or ambient air. The lack of response to changes in oxygen tension in Tet2-/- HSCs was associated with similar changes in self-renewal and quiescence genes among WT HSC-physioxia, Tet2-/- HSC-physioxia and Tet2-/- HSC-air. We define a novel molecular program involving Tet2 in regulating HSCs under physioxia.

Deficits in our understanding of natural killer cell development in mouse and human.

PURPOSE OF REVIEW: Natural killer (NK) cells are a type of immune cell that play a crucial role in the defense against cancer and viral infections. The development and maturation of NK cells is a complex process, involving the coordination of various signaling pathways, transcription factors, and epigenetic modifications. In recent years, there has been a growing interest in studying the development of NK cells. In this review, we discuss the field's current understanding of the journey a hematopoietic stem cell takes to become a fully mature NK cell and detail the sequential steps and regulation of conventional NK leukopoiesis in both mice and humans. RECENT FINDINGS: Recent studies have highlighted the significance of defining NK development stages. Several groups report differing schema to identify NK cell development and new findings demonstrate novel ways to classify NK cells. Further investigation of NK cell biology and development is needed, as multiomic analysis reveals a large diversity in NK cell development pathways. SUMMARY: We provide an overview of current knowledge on the development of NK cells, including the various stages of differentiation, the regulation of development, and the maturation of NK cells in both mice and humans. A deeper understanding of NK cell development has the potential to provide insights into new therapeutic strategies for the treatment of diseases such as cancer and viral infections.

Pharmacological inhibition of Carbonic Anhydrase IX and XII to enhance targeting of acute myeloid leukaemia cells under hypoxic conditions.

Acute myeloid leukaemia (AML) is an aggressive form of blood cancer that carries a dismal prognosis. Several studies suggest that the poor outcome is due to a small fraction of leukaemic cells that elude treatment and survive in specialised, oxygen (O2 )-deprived niches of the bone marrow. Although several AML drug targets such as FLT3, IDH1/2 and CD33 have been established in recent years, survival rates remain unsatisfactory, which indicates that other, yet unrecognized, mechanisms influence the ability of AML cells to escape cell death and to proliferate in hypoxic environments. Our data illustrates that Carbonic Anhydrases IX and XII (CA IX/XII) are critical for leukaemic cell survival in the O2 -deprived milieu. CA IX and XII function as transmembrane proteins that mediate intracellular pH under low O2 conditions. Because maintaining a neutral pH represents a key survival mechanism for tumour cells in O2 -deprived settings, we sought to elucidate the role of dual CA IX/XII inhibition as a novel strategy to eliminate AML cells under hypoxic conditions. Our findings demonstrate that the dual CA IX/XII inhibitor FC531 may prove to be of value as an adjunct to chemotherapy for the treatment of AML.

Numbers of long-term hematopoietic stem cells from bone marrow of fanca and fancc knockout mice can be greatly enhanced by their collection and processing in physioxia conditions.

Fanconi anemia (FA) is associated with bone marrow failure. Bone marrow (BM) from patients with FA and fanca-/- and fancc-/- mice are deficient in hematopoietic stem (HSCs) and progenitor cells (HPCs). Decreased HSCs/HPCs compromise their use in human and mouse hematopoietic cell transplantation (HCT) and gene therapy to correct genetic defects causing FA. We reported increased collection of HSCs from mouse bone marrow and mobilized peripheral blood, and human cord blood of normal donors after collection/processing in low (3%) oxygen (physioxia). We assessed comparative contents of long-term (LT)-HSCs from BM of fanca-/- and fancc-/- when collected/processed at 3% O2, in order to negate effects of extra physiological shock stress (EPHOSS) induced by collection/processing in ambient air. Collection/processing of BM from fanca-/- and fancc-/- mice in physioxia demonstrated a ≥3-fold increase in LT-HSCs compared to that in ambient air. This was associated with decreased phenotypic multipotential progenitor cells and functional granulocyte macrophage, erythroid, and multi-potential progenitors, results similar to that for BM from normal donor mice. Increased collection of HSCs could have clinical applicability for gene therapy and HCT.

Enhanced collection of phenotypic and engrafting human cord blood hematopoietic stem cells at 4°C.

The number of hematopoietic stem cells (HSCs) collected in cord blood (CB) at the birth of a baby is a limiting factor for efficacious use of CB in hematopoietic cell transplantation (HCT). We now demonstrate that collecting and processing of human CB at 4°C within minutes of the baby's birth results in significantly enhanced numbers of rigorously defined phenotypic HSC and self-renewing NSG immune-deficient mouse engrafting and SCID-repopulating cells. This was associated with decreased numbers of hematopoietic progenitor cells (HPC), as noted previously for hypoxia collected/processed cells blocking ambient air induced differentiation of HSC to HPC. We have thus defined a simple, cost-effective, means to collect increased numbers of CB HSC, of potential use for clinical CB HCT.

The IL-33 Receptor/ST2 acts as a positive regulator of functional mouse bone marrow hematopoietic stem and progenitor cells.

There is a paucity of information on a potential role for the IL-33 receptor/ST2 in the regulation of mouse bone marrow (BM) hematopoietic stem (HSC) and progenitor (HPC) cells. Comparing the BM of st2-/- and wild type (WT) control mice using functional assays, it was found that st2-/- BM cells had poorer engrafting capacity than WT BM in a competitive repopulating assay using congenic mice, with no changes in reconstitution of B-, T- and myeloid cells following transplantation. The BM of st2-/- mice also had fewer granulocyte-macrophage, erythroid, and multipotential progenitors than that of WT BM and these st2-/- HPC were in a slow cycling state compared to that of the rapidly cycling HPC of the WT mice. While functional assessment of HSC and HPC demonstrated that ST2 has a positive influence on regulation of HSC, we could not pick up differences in st2-/- compared to WT BM using only phenotypic analysis of HSC and HPC populations prior to transplantation, again demonstrating that phenotypic analysis of HSC and HPC do not always recapitulate the functional assessments of these immature hematopoietic cells.

Effects of Eupalinilide E and UM171, alone and in combination on cytokine stimulated ex-vivo expansion of human cord blood hematopoietic stem cells.

Eupalinilide E was assessed for ex-vivo expansion activity on hematopoietic stem cells (HSCs) from human cord blood (CB) CD34+ cells in serum-free, SCF, TPO and FL stimulated 7 day cultures. Eupalinilide E ex-vivo enhanced phenotyped (p) HSCs and glycolysis of CD34+ cells isolated 7 days after culture as measured by extracellular acidification rate, but did not alone show enhanced NSG engrafting capability of HSCs as determined by chimerism and numbers of SCID Repopulating cells, a quantitative measure of functional human HSCs. This is another example of pHSCs not necessarily recapitulating functional activity of these cells. Lack of effect on engrafting HSCs may be due to a number of possibilities, including down regulation of CXCR4 or of the homing capacity of these treated cells. However, Eupalinilide did act in an additive to synergistic fashion with UM171 to enhance ex vivo expansion of both pHSCs, and functionally engrafting HSCs. While reasons for the disconnect between pHSC and function of HSCs with Eupalinilide E alone cultured CB CD34+ cells is yet to be determined, the data suggest possible future use of Eupalinilide and UM171 together to enhance ex vivo production of CB HSCs for clinical hematopoietic cell transplantation.

Phosphatidylinositol transfer proteins regulate megakaryocyte TGF-ß1 secretion and hematopoiesis in mice.

We hypothesized that megakaryocyte (MK) phosphoinositide signaling mediated by phosphatidylinositol transfer proteins (PITPs) contributes to hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) regulation. Conditional knockout mice lacking PITPs specifically in MKs and platelets (pitpα-/- and pitpα-/-/ß-/-) bone marrow (BM) manifested decreased numbers of HSCs, MK-erythrocyte progenitors, and cycling HPCs. Further, pitpα-/-/ß-/- BM had significantly reduced engrafting capability in competitive transplantation and limiting dilution analysis. Conditioned media (CM) from cultured pitpα-/- and pitpα-/-/ß-/- BM MKs contained higher levels of transforming growth factor ß1 (TGF-ß1) and interleukin-4 (IL-4), among other myelosuppressive cytokines, than wild-type BM MKs. Correspondingly, BM flush fluid from pitpα-/- and pitpα-/-/ß-/- mice had higher concentrations of TGF-ß1. CM from pitpα-/- and pitpα-/-/ß-/- MKs significantly suppressed HPC colony formation, which was completely extinguished in vitro by neutralizing anti-TGF-ß antibody, and treatment of pitpα-/-/ß-/- mice in vivo with anti-TGF-ß antibodies completely reverted their defects in BM HSC and HPC numbers. TGF-ß and IL-4 synergized to inhibit HPC colony formation in vitro. Electron microscopy analysis of pitpα-/-/ß-/- MKs revealed ultrastructural defects with depleted α-granules and large, misshaped multivesicular bodies. Von Willebrand factor and thrombospondin-1, like TGF-ß, are stored in MK α-granules and were also elevated in CM of cultured pitpα-/-/ß-/- MKs. Altogether, these data show that ablating PITPs in MKs indirectly dysregulates hematopoiesis in the BM by disrupting α-granule physiology and secretion of TGF-ß1.

CD166 Engagement Augments Mouse and Human Hematopoietic Progenitor Function via Activation of Stemness and Cell Cycle Pathways.

Hematopoietic stem (HSC) and progenitor (HPC) cells are regulated by interacting signals and cellular and noncellular elements of the hematopoietic niche. We previously showed that CD166 is a functional marker of murine and human HSC and of cellular components of the murine niche. Selection of murine CD166+ engrafting HSC enriched for marrow repopulating cells. Here, we demonstrate that CD166-CD166 homophilic interactions enhance generation of murine and human HPC in vitro and augment hematopoietic function of these cells. Interactions between cultured CD166+ Lineage- Sca-1+ c-Kit+ (LSK) cells and CD166+ osteoblasts (OBs) significantly enhanced the expansion of colony-forming units (CFUs). Interactions between CD166+ LSK cells and immobilized CD166 protein generated more CFU in short-term cultures than between these cells and bovine serum albumin (BSA) or in cultures initiated with CD166- LSK cells. Similar results were obtained when LSK cells from wildtype (WT) or CD166 knockout (KO) (CD166-/- ) mice were used with immobilized CD166. Human cord blood CD34+ cells expressing CD166 produced significantly higher numbers of CFUs following interaction with immobilized CD166 than their CD166- counterparts. These data demonstrate the positive effects of CD166 homophilic interactions involving CD166 on the surface of murine and human HPCs. Single-cell RNA-seq analysis of CD150+ CD48- (signaling lymphocyte activation molecule (SLAM)) LSK cells from WT and CD166-/- mice incubated with immobilized CD166 protein revealed that engagement of CD166 on these cells activates cytokine, growth factor and hormone signaling, epigenetic pathways, and other genes implicated in maintenance of stem cell pluripotency-related and mitochondria-related signaling pathways. These studies provide tangible evidence implicating CD166 engagement in the maintenance of stem/progenitor cell function. Stem Cells 2019;37:1319-1330.

Toll-like receptor signaling in hematopoietic stem and progenitor cells.

PURPOSE OF REVIEW: The innate immune system is essential in the protection against microbial infection and facilitating tissue repair mechanisms. During these stresses, the maintenance of innate immune cell numbers through stress-induced or emergency hematopoiesis is key for our survival. One major mechanism to recognize danger signals is through the activation of Toll-like receptors (TLRs) on the surface of hematopoietic cells, including hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC), and nonhematopoietic cells, which recognize pathogen-derived or damaged-induced compounds and can influence the emergency hematopoietic response. This review explores how direct pathogen-sensing by HSC/HPC regulates hematopoiesis, and the positive and negative consequences of these signals. RECENT FINDINGS: Recent studies have highlighted new roles for TLRs in regulating HSC and HPC differentiation to innate immune cells of both myeloid and lymphoid origin and augmenting HSC and HPC migration capabilities. Most interestingly, new insights as to how acute versus chronic stimulation of TLR signaling regulates HSC and HPC function has been explored. SUMMARY: Recent evidence suggests that TLRs may play an important role in many inflammation-associated diseases. This suggests a possible use for TLR agonists or antagonists as potential therapeutics. Understanding the direct effects of TLR signaling by HSC and HPC may help regulate inflammatory/danger signal-driven emergency hematopoiesis.

Combinations of antioxidants and/or of epigenetic enzyme inhibitors allow for enhanced collection of mouse bone marrow hematopoietic stem cells in ambient air.

Hematopoietic cell transplantation (HCT) is a treatment for malignant and non-malignant disorders. However, sometimes the numbers of donor hematopoietic stem cells (HSC) are limiting, which can compromise the success of HCT. We recently published that collection and processing of mouse bone marrow (BM) and human cord blood cells in a hypoxic atmosphere of 3% O2 or in ambient air (~21% O2) in the presence of cyclosporine A yields increased numbers of HSC. We now show that collection and processing of mouse BM cells in ambient air in the presence of specific combinations of anti-oxidants and/or inhibitors of epigenetic enzymes can also enhance the collection of HSC, information of potential relevance for enhanced efficacy of HCT.

Erythropoietin modulation is associated with improved homing and engraftment after umbilical cord blood transplantation.

Umbilical cord blood (UCB) engraftment is in part limited by graft cell dose, generally one log less than that of bone marrow (BM)/peripheral blood (PB) cell grafts. Strategies toward increasing hematopoietic stem/progenitor cell (HSPC) homing to BM have been assessed to improve UCB engraftment. Despite recent progress, a complete understanding of how HSPC homing and engraftment are regulated is still elusive. We provide evidence that blocking erythropoietin (EPO)-EPO receptor (R) signaling promotes homing to BM and early engraftment of UCB CD34+ cells. A significant population of UCB CD34+ HSPC expresses cell surface EPOR. Exposure of UCB CD34+ HSPC to EPO inhibits their migration and enhances erythroid differentiation. This migratory inhibitory effect was reversed by depleting EPOR expression on HSPC. Moreover, systemic reduction in EPO levels by hyperbaric oxygen (HBO) used in a preclinical mouse model and in a pilot clinical trial promoted homing of transplanted UCB CD34+ HSPC to BM. Such a systemic reduction of EPO in the host enhanced myeloid differentiation and improved BM homing of UCB CD34+ cells, an effect that was overcome with exogenous EPO administration. Of clinical relevance, HBO therapy before human UCB transplantation was well-tolerated and resulted in transient reduction in EPO with encouraging engraftment rates and kinetics. Our studies indicate that systemic reduction of EPO levels in the host or blocking EPO-EPOR signaling may be an effective strategy to improve BM homing and engraftment after allogeneic UCB transplantation. This clinical trial was registered at www.ClinicalTrials.gov (#NCT02099266).

A role for intracellular and extracellular DEK in regulating hematopoiesis.

PURPOSE OF REVIEW: Hematopoietic stem/progenitor cell fate decision during hematopoiesis is regulated by intracellular and extracellular signals such as transcription factors, growth factors, and cell-to-cell interactions. In this review, we explore the function of DEK, a nuclear phosphoprotein, on gene regulation. We also examine how DEK is secreted and internalized by cells, and discuss how both endogenous and extracellular DEK regulates hematopoiesis. Finally, we explore what currently is known about the regulation of DEK during inflammation. RECENT FINDINGS: DEK negatively regulates the proliferation of early myeloid progenitor cells but has a positive effect on the differentiation of mature myeloid cells. Inflammation regulates intracellular DEK concentrations with inflammatory stimuli enhancing DEK expression. Inflammation-induced nuclear factor-kappa B activation is regulated by DEK, resulting in changes in the production of other inflammatory molecules such as IL-8. Inflammatory stimuli in turn regulates DEK secretion by cells of hematopoietic origin. However, how inflammation-induced expression and secretion of DEK regulates hematopoiesis remains unknown. SUMMARY: Understanding how DEK regulates hematopoiesis under both homeostatic and inflammatory conditions may lead to a better understanding of the biology of HSCs and HPCs. Furthering our knowledge of the regulation of hematopoiesis will ultimately lead to new therapeutics that may increase the efficacy of hematopoietic stem cell transplantation.

Mild Heat Treatment Primes Human CD34(+) Cord Blood Cells for Migration Toward SDF-1α and Enhances Engraftment in an NSG Mouse Model.

Simple efforts are needed to enhance cord blood (CB) transplantation. We hypothesized that short-term exposure of CD34(+) CB cells to 39.5°C would enhance their response to stromal-derived factor-1 (SDF-1), by increasing lipid raft aggregation and CXCR4 expression, thus leading to enhanced engraftment. Mild hyperthermia (39.5°C) significantly increased the percent of CD34(+) CB that migrated toward SDF-1. This was associated with increased expression of CXCR4 on the cells. Mechanistically, mild heating increased the percent of CD34(+) cells with aggregated lipid rafts and enhanced colocalization of CXCR4 within lipid raft domains. Using methyl-ß-cyclodextrin (MßCD), an agent that blocks lipid raft aggregation, it was determined that this enhancement in chemotaxis was dependent upon lipid raft aggregation. Colocalization of Rac1, a GTPase crucial for cell migration and adhesion, with CXCR4 to the lipid raft was essential for the effects of heat on chemotaxis, as determined with an inhibitor of Rac1 activation, NSC23766. Application-wise, mild heat treatment significantly increased the percent chimerism as well as homing and engraftment of CD34(+) CB cells in sublethally irradiated non-obese diabetic severe combined immunodeficiency IL-2 receptor gamma chain d (NSG) mice. Mild heating may be a simple and inexpensive means to enhance engraftment following CB transplantation in patients.

Baseline tumor growth and immune control in laboratory mice are significantly influenced by subthermoneutral housing temperature.

We show here that fundamental aspects of antitumor immunity in mice are significantly influenced by ambient housing temperature. Standard housing temperature for laboratory mice in research facilities is mandated to be between 20-26 °C; however, these subthermoneutral temperatures cause mild chronic cold stress, activating thermogenesis to maintain normal body temperature. When stress is alleviated by housing at thermoneutral ambient temperature (30-31 °C), we observe a striking reduction in tumor formation, growth rate and metastasis. This improved control of tumor growth is dependent upon the adaptive immune system. We observe significantly increased numbers of antigen-specific CD8(+) T lymphocytes and CD8(+) T cells with an activated phenotype in the tumor microenvironment at thermoneutrality. At the same time there is a significant reduction in numbers of immunosuppressive MDSCs and regulatory T lymphocytes. Notably, in temperature preference studies, tumor-bearing mice select a higher ambient temperature than non-tumor-bearing mice, suggesting that tumor-bearing mice experience a greater degree of cold-stress. Overall, our data raise the hypothesis that suppression of antitumor immunity is an outcome of cold stress-induced thermogenesis. Therefore, the common approach of studying immunity against tumors in mice housed only at standard room temperature may be limiting our understanding of the full potential of the antitumor immune response.

Ames hypopituitary dwarf mice demonstrate imbalanced myelopoiesis between bone marrow and spleen.

Ames hypopituitary dwarf mice are deficient in growth hormone, thyroid-stimulating hormone, and prolactin. The phenotype of these mice demonstrates irregularities in the immune system with skewing of the normal cytokine milieu towards a more anti-inflammatory environment. However, the hematopoietic stem and progenitor cell composition of the bone marrow (BM) and spleen in Ames dwarf mice has not been well characterized. We found that there was a significant decrease in overall cell count when comparing the BM and spleen of 4-5 month old dwarf mice to their littermate controls. Upon adjusting counts to differences in body weight between the dwarf and control mice, the number of granulocyte-macrophage progenitors, confirmed by immunophenotyping and colony-formation assay was increased in the BM. In contrast, the numbers of all myeloid progenitor populations in the spleen were greatly reduced, as confirmed by colony-formation assays. This suggests that there is a shift of myelopoiesis from the spleen to the BM of Ames dwarf mice; however, this shift does not appear to involve erythropoiesis. The reasons for this unusual shift in spleen to marrow hematopoiesis in Ames dwarf mice are yet to be determined but may relate to the decreased hormone levels in these mice.

Granzyme B-mediated damage of CD8+ T cells impairs graft-versus-tumor effect.

Allogeneic hematopoietic cell transplantation is an established treatment for hematologic and other malignancies. Donor-derived immune cells can identify and attack host tumor cells, producing a graft-versus-tumor (GVT) effect that is crucial to the treatment. Using multiple tumor models and diverse donor-host combinations, we have studied the role of granzyme B (GzmB) in GVT effect. We first confirmed previous findings that GzmB deficiency diminished the ability of a high dose of CD8(+) T cells to cause lethal graft-versus-host disease. However, when GVT studies were performed using a moderate cell dose that the hosts could tolerate, GzmB(-/-) CD8(+) T cells demonstrated a significantly enhanced GVT effect. GzmB-mediated, activation-induced cell death in wild-type CD8(+) T cells was found responsible for their reduced GVT activity. Conversely, GzmB(-/-) CD8(+) T cells exhibited enhanced expansion, skewed toward an effector or effector memory phenotype, and produced higher amounts of IFN-γ and Fas ligand that might contribute to GzmB-independent tumor control. These findings demonstrate for the first time, to our knowledge, that GzmB-mediated damage of CD8(+) T cells impairs the desired GVT effect. This study suggests that inhibiting donor-derived GzmB function may represent a promising strategy to improve GVT effect without exacerbating graft-versus-host disease.

Elevating body temperature enhances hematopoiesis and neutrophil recovery after total body irradiation in an IL-1-, IL-17-, and G-CSF-dependent manner.

Neutropenia is a common side effect of cytotoxic chemotherapy and radiation, increasing the risk of infection in these patients. Here we examined the impact of body temperature on neutrophil recovery in the blood and bone marrow after total body irradiation (TBI). Mice were exposed to either 3 or 6 Gy TBI followed by a mild heat treatment that temporarily raised core body temperature to approximately 39.5°C. Neutrophil recovery was then compared with control mice that received either TBI alone heat treatment alone. Mice that received both TBI and heat treatment exhibited a significant increase in the rate of neutrophil recovery in the blood and an increase in the number of marrow hematopoietic stem cells and neutrophil progenitors compared with that seen in mice that received either TBI or heat alone. The combination treatment also increased G-CSF concentrations in the serum, bone marrow, and intestinal tissue and IL-17, IL-1ß, and IL-1α concentrations in the intestinal tissue after TBI. Neutralizing G-CSF or inhibiting IL-17 or IL-1 signaling significantly blocked the thermally mediated increase in neutrophil numbers. These findings suggest that a physiologically relevant increase in body temperature can accelerate recovery from neutropenia after TBI through a G-CSF-, IL-17-, and IL-1-dependent mechanism.

Concise review: role of DEK in stem/progenitor cell biology.

Understanding the factors that regulate hematopoiesis opens up the possibility of modifying these factors and their actions for clinical benefit. DEK, a non-histone nuclear phosphoprotein initially identified as a putative proto-oncogene, has recently been linked to regulate hematopoiesis. DEK has myelosuppressive activity in vitro on proliferation of human and mouse hematopoietic progenitor cells and enhancing activity on engraftment of long-term marrow repopulating mouse stem cells, has been linked in coordinate regulation with the transcription factor C/EBPα, for differentiation of myeloid cells, and apparently targets a long-term repopulating hematopoietic stem cell for leukemic transformation. This review covers the uniqueness of DEK, what is known about how it now functions as a nuclear protein and also as a secreted molecule that can act in paracrine fashion, and how it may be regulated in part by dipeptidylpeptidase 4, an enzyme known to truncate and modify a number of proteins involved in activities on hematopoietic cells. Examples are provided of possible future areas of investigation needed to better understand how DEK may be regulated and function as a regulator of hematopoiesis, information possibly translatable to other normal and diseased immature cell systems.

Protocol for enrichment and functional analysis of human hematopoietic cells from umbilical cord blood.

Umbilical cord blood (CB) is a donor source for hematopoietic cell therapies. Understanding what drives hematopoietic stem and progenitor cell function is critical to our understanding of the usage of CB in hematopoietic cell therapies. Here, we describe how to isolate and analyze the function of human hematopoietic cells from umbilical CB. This protocol demonstrates assays that measure phenotypic properties and hematopoietic cell potency. For complete details on the use and execution of this protocol, please refer to Broxmeyer et al.1.