Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock.

Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.

Dipeptidyl Peptidase 4 Inhibition for Prophylaxis of Acute Graft-versus-Host Disease.

BACKGROUND: Dipeptidyl peptidase 4 (DPP-4; also known as CD26), a transmembrane receptor expressed on T cells, has a costimulatory function in activating T cells. In a mouse model, down-regulation of CD26 prevented graft-versus-host disease (GVHD) but preserved graft-versus-tumor effects. Whether inhibition of DPP-4 with sitagliptin may prevent acute GVHD after allogeneic stem-cell transplantation is not known. METHODS: We conducted a two-stage, phase 2 clinical trial to test whether sitagliptin plus tacrolimus and sirolimus would reduce the incidence of grade II to IV acute GVHD from 30% to no more than 15% by day 100. Patients received myeloablative conditioning followed by mobilized peripheral-blood stem-cell transplants. Sitagliptin was given orally at a dose of 600 mg every 12 hours starting the day before transplantation until day 14 after transplantation. RESULTS: A total of 36 patients who could be evaluated, with a median age of 46 years (range, 20 to 59), received transplants from matched related or unrelated donors. Acute GVHD occurred in 2 of 36 patients by day 100; the incidence of grade II to IV GVHD was 5% (95% confidence interval [CI], 1 to 16), and the incidence of grade III or IV GVHD was 3% (95% CI, 0 to 12). Nonrelapse mortality was zero at 1 year. The 1-year cumulative incidences of relapse and chronic GVHD were 26% (95% CI, 13 to 41) and 37% (95% CI, 22 to 53), respectively. GVHD-free, relapse-free survival was 46% (95% CI, 29 to 62) at 1 year. Toxic effects were similar to those seen in patients undergoing allogeneic stem-cell transplantation. CONCLUSIONS: In this nonrandomized trial, sitagliptin in combination with tacrolimus and sirolimus resulted in a low incidence of grade II to IV acute GVHD by day 100 after myeloablative allogeneic hematopoietic stem-cell transplantation. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT02683525.).

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.

The neurotransmitter receptor Gabbr1 regulates proliferation and function of hematopoietic stem and progenitor cells.

Hematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the γ-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry and found that the endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1-knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared with wild-type littermates. Moreover, Gabbr1-null hematopoietic stem cells had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1-null HSPCs were less proliferative under steady-state conditions and upon stress. Colony-forming unit assays demonstrated that almost all Gabbr1-null HSPCs were in a slow or noncycling state. In vitro differentiation of Gabbr1-null HSPCs in cocultures produced fewer overall cell numbers with significant defects in differentiation and expansion of the B-cell lineage. To determine whether a GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared with GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.

Leukemia Inhibitory Factor Promotes Survival of Hematopoietic Progenitors Ex Vivo and Is Post-Translationally Regulated by DPP4.

Hematopoietic cells are regulated in part by extracellular cues from cytokines. Leukemia inhibitory factor (LIF) promotes survival, self-renewal, and pluripotency of mouse embryonic stem cells (mESC). While genetic deletion of LIF affects hematopoietic progenitor cells (HPCs), the direct effect of LIF protein exposure on HPC survival is not known. Furthermore, post-translational modifications (PTM) of LIF and their effects on its function have not been evaluated. We demonstrate that treatment with recombinant LIF preserves mouse and human HPC numbers in stressed conditions when growth factor addition is delayed ex vivo. We show that Lif is upregulated in response to irradiation-induced stress. We reveal novel PTM of LIF where it is cleaved twice by dipeptidyl peptidase 4 (DPP4) protease so that it loses its 4 N-terminal amino acids. This truncation of LIF down-modulates LIF's ability to preserve functional HPC numbers ex vivo following delayed growth factor addition. DPP4-truncated LIF blocks the ability of full-length LIF to preserve functional HPC numbers. This LIF role and its novel regulation by DPP4 have important implications for normal and stress hematopoiesis, as well as for other cellular contexts in which LIF and DPP4 are implicated.

Ex Vivo Expansion and Homing of Human Cord Blood Hematopoietic Stem Cells.

Cord blood (CB) has been proven to be an alternative source of haematopoietic stem cells (HSCs) for clinical transplantation and has multiple advantages, including but not limited to greater HLA compatibility, lower incidence of graft-versus-host disease (GvHD), higher survival rates and lower relapse rates among patients with minimal residual disease. However, the limited number of HSCs in a single CB unit limits the wider use of CB in clinical treatment. Many efforts have been made to enhance the efficacy of CB HSC transplantation, particularly by ex vivo expansion or enhancing the homing efficiency of HSCs. In this chapter, we will document the major advances regarding human HSC ex vivo expansion and homing and will also discuss the possibility of clinical translation of such laboratory work.

Leukaemogenic effects of Ptpn11 activating mutations in the stem cell microenvironment.

Germline activating mutations of the protein tyrosine phosphatase SHP2 (encoded by PTPN11), a positive regulator of the RAS signalling pathway, are found in 50% of patients with Noonan syndrome. These patients have an increased risk of developing leukaemia, especially juvenile myelomonocytic leukaemia (JMML), a childhood myeloproliferative neoplasm (MPN). Previous studies have demonstrated that mutations in Ptpn11 induce a JMML-like MPN through cell-autonomous mechanisms that are dependent on Shp2 catalytic activity. However, the effect of these mutations in the bone marrow microenvironment remains unclear. Here we report that Ptpn11 activating mutations in the mouse bone marrow microenvironment promote the development and progression of MPN through profound detrimental effects on haematopoietic stem cells (HSCs). Ptpn11 mutations in mesenchymal stem/progenitor cells and osteoprogenitors, but not in differentiated osteoblasts or endothelial cells, cause excessive production of the CC chemokine CCL3 (also known as MIP-1α), which recruits monocytes to the area in which HSCs also reside. Consequently, HSCs are hyperactivated by interleukin-1ß and possibly other proinflammatory cytokines produced by monocytes, leading to exacerbated MPN and to donor-cell-derived MPN following stem cell transplantation. Remarkably, administration of CCL3 receptor antagonists effectively reverses MPN development induced by the Ptpn11-mutated bone marrow microenvironment. This study reveals the critical contribution of Ptpn11 mutations in the bone marrow microenvironment to leukaemogenesis and identifies CCL3 as a potential therapeutic target for controlling leukaemic progression in Noonan syndrome and for improving stem cell transplantation therapy in Noonan-syndrome-associated leukaemias.

Consequences of coronavirus infections for primitive and mature hematopoietic cells: new insights and why it matters.

PURPOSE OF REVIEW: In recent history there have been three outbreaks of betacoronavirus infections in humans, with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; causing Coronavirus disease 2019 [COVID-19]) outbreak leading to over two million deaths, with a rapidly rising death toll. Much remains unknown about host cells and tissues affected by coronavirus infections, including the hematopoietic system. Here, we discuss the recent findings examining effects that coronavirus infection or exposure has on hematopoietic cells and the clinical implications for these effects. RECENT FINDINGS: Recent studies have centered on SARS-CoV-2, demonstrating that hematopoietic stem and progenitor cells and mature immune cells may be susceptible to infection and are impacted functionally by exposure to SARS-CoV-2 Spike protein. These findings have important implications regarding hematologic complications arising from COVID-19 and other coronavirus-induced disease, which we discuss here. SUMMARY: Infection with coronaviruses sometimes leads to hematologic complications in patients, and these hematologic complications are associated with poorer prognosis. These hematologic complications may be caused by coronavirus direct infection or impact on primitive hematopoietic cells or mature immune cells, by indirect effects on these cells, or by a combination thereof. It is important to understand how hematologic complications arise in order to seek new treatments to improve patient outcomes.

Expression of gilt acts as a positive regulator of mouse hematopoietic progenitor cells.

Gamma interferon inducible lysosomal thiol reductase (GILT), is known to be involved in immunity, but its role in hematopoiesis has not been previously reported. Herein, we demonstrate using gilt knockout (-/-) mice that loss of gilt associates with decreased numbers and cycling status of femoral hematopoietic progenitor cells (CFU-GM, BFU-E, and CFU-GEMM) with more modest effects on splenic progenitor cells. Thus, GILT is associated with positive regulation of hematopoietic progenitor cells in mice, mainly in bone marrow.

CXCL15/Lungkine has suppressive activity on proliferation and expansion of multi-potential, erythroid, granulocyte and macrophage progenitors in S-phase specific manner.

Cytokines/chemokines regulate hematopoiesis, most having multiple cell actions. Numerous but not all chemokine family members act as negative regulators of hematopoietic progenitor cell (HPC) proliferation, but very little is known about such effects of the chemokine, CXCL15/Lungkine. We found that CXCL15/Lungkine-/- mice have greatly increased cycling of multi cytokine-stimulated bone marrow and spleen hematopoietic progenitor cells (HPCs: CFU-GM, BFU-E, and CFU-GEMM) and CXCL15 is expressed in many bone marrow progenitor and other cell types. This suggests that CXCL15/Lungkine acts as a negative regulator of the cell cycling of these HPCs in vivo. Recombinant murine CXCL15/Lungkine, decreased numbers of functional HPCs during cytokine-enhanced ex-vivo culture of lineage negative mouse bone marrow cells. Moreover, CXCL15/Lungkine, through S-Phase specific actions, was able to suppress in vitro colony formation of normal wildtype mouse bone marrow CFU-GM, CFU-G, CFU-M, BFU-E, and CFU-GEMM. This clearly identifies the negative regulatory activity of CXCL15/Lungkine on proliferation of multiple types of mouse HPCs.

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.

Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells.

Understanding physiologic T-cell development from hematopoietic stem (HSCs) and progenitor cells (HPCs) is essential for development of improved hematopoietic cell transplantation (HCT) and emerging T-cell therapies. Factors in the thymic niche, including Notch 1 receptor ligand, guide HSCs and HPCs through T-cell development in vitro. We report that physiologically relevant oxygen concentration (5% O2 , physioxia), an important environmental thymic factor, promotes differentiation of cord blood CD34+ cells into progenitor T (proT) cells in serum-free and feeder-free culture system. This effect is enhanced by a potent reducing and antioxidant agent, ascorbic acid. Human CD34+ cell-derived proT cells in suspension cultures maturate into CD3+ T cells in an artificial thymic organoid (ATO) culture system more efficiently when maintained under physioxia, compared to ambient air. Low oxygen tension acts as a positive regulator of HSC commitment and HPC differentiation toward proT cells in the feeder-free culture system and for further maturation into T cells in the ATO. Culturing HSCs/HPCs in physioxia is an enhanced method of effective progenitor T and mature T-cell production ex vivo and may be of future use for HCT and T-cell immunotherapies.

An expanded role for dipeptidyl peptidase 4 in cell regulation.

PURPOSE OF REVIEW: Dipeptidyl peptidase 4 (DPP4) is a serine protease with diverse regulatory functions in healthy and diseased cells. Much remains unknown about the mechanisms and targets of DPP4. Here we discuss new studies exploring DPP4-mediated cellular regulation, provide an updated list of potential targets of DPP4, and discuss clinical implications of each. RECENT FINDINGS: Recent studies have sought enhanced efficacy of targeting DPP4's role in regulating hematopoietic stem and progenitor cells for improved clinical application. Further studies have identified DPP4 functions in different cellular compartments and have proposed ways to target this protein in malignancy. These findings, together with an expanded list of putative extracellular, cell surface, and intracellular DPP4 targets, provide insight into new DPP4-mediated cell regulation. SUMMARY: DPP4 posttranslationally modifies proteins and peptides with essential roles in hematopoietic cell regulation, stem cell transplantation, and malignancy. Targets include secreted signaling factors and may include membrane proteins and transcription factors critical for different hematopoietic functions. Knowing these targets and functions can provide insight into new regulatory roles for DPP4 that may be targeted to enhance transplantation, treat disease, and better understand different regulatory pathways of hematopoiesis.

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.

Erythropoietin surprises: an immune saga.

Erythropoietin (EPO) an erythropoietic stimulating agent also exerts effects on other cell systems. Nairz et al. (2011) now link EPO and intracellular signaling through the EPO receptor (EPOR) to innate immune cell activity via macrophages.

Progress towards improving homing and engraftment of hematopoietic stem cells for clinical transplantation.

PURPOSE OF REVIEW: Hematopoietic cell transplantation (HCT) is a life-saving treatment for a variety of hematological and nonhematological disorders. Successful clinical outcomes after transplantation rely on adequate hematopoietic stem cell (HSC) numbers, and the homing and subsequent short-term and long-term engraftment of these cells in the bone marrow. Enhancing the homing capability of HSCs has the potential for high impact on improving HCT and patient survival. RECENT FINDINGS: There are a number of ways to enhance HSC engraftment. Neutralizing negative epigenetic regulation by histone deacetylase 5 (HDAC5) increases surface CXCR4 expression and promotes human HSC homing and engraftment in immune-deficient NSG (NOD.Cg-Prkdc IL2rgt/Sz) mice. Short-term treatment of cells with glucocorticoids, pharmacological stabilization of hypoxia-inducible factor (HIF)-1α, increasing membrane lipid raft aggregation, and inhibition of dipeptidyl peptidase 4 (DPP4) facilitates HSC homing and engraftment. Added to these procedures, modulating the mitochondria permeability transition pore (MPTP) to mitigate ambient air-induced extra physiological oxygen stress/shock (EPHOSS) by hypoxic harvest and processing, or using cyclosporine A during air collection increases functional HSC numbers and improves HSC engraftment. SUMMARY: A better understanding of the regulation of human HSC homing mediated by various signaling pathways will facilitate development of more efficient means to enhance HCT efficacy.