Stem Cell Factor Production Research Articles

PAR‐2 is involved in melanogenesis by mediating stem cell factor production in keratinocytes

<scp>PAR</scp>‐2 is involved in melanogenesis by mediating stem cell factor production in keratinocytes

The lysophosphatidic acid receptor LPA4 regulates hematopoiesis-supporting activity of bone marrow stromal cells.

Lysophosphatidic acid (LPA) is a pleiotropic lipid mediator that acts through G protein-coupled receptors (LPA1-6). Although several biological roles of LPA4 are becoming apparent, its role in hematopoiesis has remained unknown. Here, we show a novel regulatory role for LPA4 in hematopoiesis. Lpar4 mRNA was predominantly expressed in mouse bone marrow (BM) PDGFRα+ stromal cells, known as the components of the hematopoietic stem/progenitor cell (HSPC) niche. Compared with wild-type mice, LPA4-deficient mice had reduced HSPC numbers in the BM and spleen and were hypersusceptible to myelosuppression, most likely due to impairments in HSPC recovery and stem cell factor production in the BM. Analysis of reciprocal BM chimeras (LPA4-deficient BM into wild-type recipients and vice versa) indicated that stromal cells likely account for these phenotypes. Consistently, LPA4-deficient BM stromal cells showed downregulated mRNA expression of stem cell factor and tenascin-c in vitro. Taken together, these results suggest a critical and novel role for the LPA/LPA4 axis in regulating BM stromal cells.

Interleukin-1 beta induces the expression and production of stem cell factor by epithelial cells: crucial involvement of the PI-3K/mTOR pathway and HIF-1 transcription complex.

Potential crosslinks between inflammation and leukaemia have been discussed for some time, but experimental evidence to support this dogma is scarce. In particular, it is important to understand the mechanisms responsible for potential upregulation of proto-oncogenic growth factor expressions by inflammatory mediators. Here, we investigated the ability of the highly inflammatory cytokine interleukin-1 beta (IL-1β) to induce the production of stem cell factor (SCF), which is a major hematopoietic growth factor that controls the progression of acute myeloid leukaemia upon malignant transformation of haematopoietic myeloid cells. We found that human IL-1β induced the expression/secretion of SCF in MCF-7 human epithelial breast cancer cells and that this process depended on the hypoxia-inducible factor 1 (HIF-1) transcription complex. We also demonstrated a crucial role of the phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway in IL-1β-induced HIF-1α accumulation in MCF-7 cells. Importantly, mTOR was also found to play a role in IL-1β-induced SCF production. Furthermore, a tendency for a positive correlation of IL-1β and SCF levels in the plasma of healthy human donors was observed. Altogether, our results demonstrate that IL-1β, which normally bridges innate and adaptive immunity, induces the production of the major haematopoietic/proleukaemic growth factor SCF through the PI-3K/mTOR pathway and the HIF-1 transcription complex. These findings strongly support a cross-talk between inflammation and acute myeloid leukaemia.

C-Kit signaling determines neointimal hyperplasia in arteriovenous fistulae.

Stenosis of arteriovenous (A-V) fistulae secondary to neointimal hyperplasia (NIH) compromises dialysis delivery, which worsens patients' quality of life and increases medical costs associated with the maintenance of vascular accesses. In the present study, we evaluated the role of the receptor tyrosine kinase c-Kit in A-V fistula neointima formation. Initially, c-Kit was found in the neointima and adventitia of human brachiobasilic fistulae, whereas it was barely detectable in control veins harvested at the time of access creation. Using the rat A-V fistula model to study venous vascular remodeling, we analyzed the spatial and temporal pattern of c-Kit expression in the fistula wall. Interestingly, c-Kit immunoreactivity increased with time after anastomosis, which concurred with the accumulation of cells in the venous intima. In addition, c-Kit expression in A-V fistulae was positively altered by chronic kidney failure conditions. Both blockade of c-Kit with imatinib mesylate (Gleevec) and inhibition of stem cell factor production with a specific short hairpin RNA prevented NIH in the outflow vein of experimental fistulae. In agreement with these data, impaired c-Kit activity compromised the development of NIH in A-V fistulae created in c-KitW/Wv mutant mice. These results suggest that targeting of the c-Kit signaling pathway may be an effective approach to prevent postoperative NIH in A-V fistulae.

Cxcl12 production by bone marrow Nes+ MSCs is downregulated in malnourished mice

Hematopoietic stem cells (HSCs) mainly reside in the bone marrow (BM) where signals generated by their niche regulate their self-renewal, proliferation and trafficking. Key components of the perivascular niche include endothelial cells, mesenchymal progenitors and CXCL12-expressing mesechymal cells - including CAR cells, leptin receptor+ (LepR+) cells and Nestin+ (Nes+) cells. These cells produce several factors that have been implicated in HSC maintenance, including CXCL12, stem cell factor (SCF) and others. CXCL12 plays a crucial role in maintaining HSC function, including retention in the bone marrow and quiescence. On the other hand, SCF contributes to the self-renewal of HSCs. Protein-energy malnutrition (PEM) is associated to hematopoietic failure, per BM hypoplasia, HSCs primarily in a quiescent stage and an impairment of BM microenvironment. Since mesenchymal stem cells (MSC) and endothelial cells are critical to regulate HSCs in the BM microenvironment, we hypothesized that PEM could affect the CXCL12 and SCF production. To test this assumption, we evaluated the possible effects of PEM on BM MSC and endothelial-like MSC (EL-MSC) through the production of CXCL12 and SCF. C57BL/6 mice were induced to PEM by a 2% protein-diet. BM MSCs were isolated and differentiated into EL-MSC and Nes, LepR, CXCL12 and SCF mRNA expression were quantified as well as the production of CXCL12 and SCF by ELISA. We observed that all cells studied were Nes+, however EL-MSCs from control and PEM groups showed higher LepR mRNA expression. The production of SCF by MSC from malnourished animals was increased in comparison to control MSC, although in EL-MSC from malnourished mice, SCF production was decreased when compared to control EL-MSC. Concerning the CXCL12 production by MSC and EL-MSCs, in PEM it was downregulated in comparison to control group. These results suggest that, as HSC pool is reduced in PEM, there is an unsuccessfully attempt to provide HSC pool expansion on malnourished mice.

Expression and possible role of IGF-IR in the mouse gastric myenteric plexus and smooth muscles

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) have tremendous trophic effects on the central, peripheral and enteric neurons. The loss of IGF-IR contributes to the development of diabetic gastroparesis. However, the nature and the function of the IGF-IR+ cells in the gastric myenteric plexus remain unclear. In this study, anti-ChAT, anti-S100β or anti-c-KIT antibodies were used to co-label IGF-IR+ cells and neurons, glial cells or interstitial cells of Cajal (ICCs), respectively. We also generated type 1 diabetic mice (DM) to explore the influence of impaired IGF-I/IGF-IR in the myenteric neurons. Results showed that IGF-IR was expressed in the epithelium, smooth muscles and myenteric plexi of the mouse stomach. Most of the IGF-IR+ cells in the myenteric plexi were ChAT+ cholinergic neurons, but not enteric glial cells and there were more IGF-IR+ neurons and fibers in the gastric antrum than in the corpus. The IGF-IR+/ChAT+ neurons and ICCs were closely juxtaposed, but distinctly distributed in the myenteric plexus, indicating a possible role for the IGF-IR+/ChAT+ neurons in the mediation of gastric motility through ICCs. Moreover, the decrease of IGF-IR and cholinergic neurons in the myenteric plexi and smooth muscles of DM mice suggested that IGF-I/IGF-IR signaling might play a role in neuron survival and neurite outgrowth, as well as stem cell factor (SCF) production, which is required for the development of ICCs. Our results provide insights into the effects of IGF-I/IGF-IR signaling on the development of gastrointestinal motility disorders.

The expression of cannabinoid receptor 1 is significantly increased in atopic patients

The expression of cannabinoid receptor 1 is significantly increased in atopic patients

Production of stem cell factor in canine mast cell tumors

Mast cell tumor (MCT) is the most common cutaneous tumor in dogs. We recently revealed that production of stem cell factor (SCF) contributes to the proliferation of neoplastic mast cells in an autocrine/paracrine manner. The aim of the present study was to determine the contribution of the mechanism in clinical MCTs. In consequence, high SCF expression (>10 times compared to HRMC cells) was observed in 5 of 7 MCT samples used in the study regardless of KIT mutation, which was confirmed in immunohistochemical analysis. In addition, production of SCF was observed in Ki-67-positive cells in the MCT xenograft. These results indicate the broad contribution of SCF autocrine/paracrine mechanism on clinical MCTs, providing the rationale for the clinical use of KIT inhibitors regardless of KIT mutation.

PTU-110 Circulating Bone Marrow-Derived Stem Cells and Stem Cell Factor Serum Level in Chronic Hepatitis C: Relation to Hepatic Proliferation and Fibrosis

IntroductionBone marrow-derived stem cells (BMSCs) are pluripotent cells that can be mobilised into circulation and recruited to sites of inflammation where they promote local tissue repair. Therefore, the present work...

Paracrine cytokine mechanisms underlying the hyperpigmentation of seborrheic keratosis in covered skin areas

We previously reported that increased expression of the endothelin (EDN)1/EDNB receptor (EDNBR) as well as the stem cell factor (SCF)/SCF receptor (c-KIT) linkages is mainly responsible for the activation of melanocytes in the epidermal hyperpigmentation of ultraviolet (UV)-B melanosis and lentigo senilis (LS). In this study, we characterized seborrheic keratosis (SK) to examine the paracrine cytokine mechanism(s) involved in its epidermal hyperpigmentation by reverse transcription polymerase chain reaction, immunohistochemistry and western blotting analyses. In contrast to our previous study which showed the upregulated expression of EDN1 and EDNBR at the transcriptional and translational levels in the epidermis of SK, we observed unexpectedly that the cytokine SCF and its receptor c-KIT are not upregulated, but are downregulated at both the gene and protein levels. We established SK cell lines to examine whether SK basaloid cells are less sensitive to SCF-inducible stimulation than are normal human keratinocytes (NHK). Comparison of the stimulatory effects of interleukin (IL)-1α or tumor necrosis factor (TNF)-α on SCF production between SK cells and NHK demonstrated that SK cells do not respond to IL-1α or TNF-α to stimulate production of SCF, whereas a significant stimulation of SCF is elicited by those same cytokines in NHK. These finding underscore a role of phenotypic changes in melanogenic cytokine production in the epidermis between SK and LS/UV-B melanosis.

Stem cell factor expression in B cell malignancies is influenced by the niche

Our group has previously demonstrated that expression of the cytokine stem cell factor (SCF) by leukemic blasts is a frequent finding in pre-B acute lymphoblastic leukemia (ALL). Whether SCF expression is a feature of other B cell malignancies and whether cross-talk from the local microenvironment modulates malignant cell SCF production are, however, unknown. Here we show using immunohistochemistry that SCF is expressed by a wide variety of indolent and aggressive B cell malignancies involving the bone marrow (BM) or lymph nodes (LNs). In diseases such as follicular lymphoma (FL), however, where lymphoma cells uniquely associate with the BM endosteal niche, BM lymphoma does not express SCF, while LN involvement is SCF positive. In contrast, cases of FL with high-grade transformation in the BM are SCF positive. These data suggest that lymphoma cell interaction with the endosteal niche inhibits SCF production, and that FL cells become independent of this microenvironment effect following transformation.

A high-fat diet increases interleukin-3 and granulocyte colony-stimulating factor production by bone marrow cells and triggers bone marrow hyperplasia and neutrophilia in wistar rats

It is well established that the excessive consumption of a high-fat diet (HFD) results in overweight, obesity and an increase in leptin concentrations, which triggers a chronic inflammatory condition that is associated with a high white blood cell count. Two-month-old male Wistar rats were fed a control (CON) diet or an HFD for 12 weeks. After this period, hemogram, myelogram and biochemical parameters were evaluated along with the cell cycle and the percentage of CD34(+) cells in the bone marrow as well as cell proliferation and differentiation assays and the production of stem cell factor, interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF). The HFD animals exhibited leukocytosis and neutrophilia with increased C-reactive protein, leptin, cholesterol and triglyceride concentrations. In the HFD group, the bone marrow revealed myeloid hyperplasia, especially of the granulocytic compartment with a higher percentage of CD34(+) cells and a higher percentage of cells in the G2/S/M cell cycle phases. In addition, the HFD bone marrow cells had a higher capacity to proliferate and differentiate into granulocytic cells in an in vitro system and a higher capacity to produce IL-3 and G-CSF. These data led us to infer that the HFD induces leukocytosis and neutrophilia suggesting alterations in hematopoiesis system modulation.

Cannabinoid receptor 1 controls human mucosal-type mast cell degranulation and maturation in situ

Because many chronic inflammatory and allergic disorders are intimately linked to excessive mast cell (MC) numbers and activation, it is clinically important to understand the physiologic mechanisms preventing excess MC accumulation/degranulation in normal human tissues. Because endocannabinoids are increasingly recognized as neuroendocrine regulators of MC biology, we investigated how cannabinoid receptor (CB) 1 signaling affects human mucosal-type mast cells (hMMCs). Using organ-cultured nasal polyps as a surrogate tissue for human bronchial mucosa, we investigated how CB1 stimulation, inhibition, or knockdown affects hMMC biology using quantitative (immuno)histomorphometry and electron microscopy. Kit(+) hMMCs express functional CB1 in situ. Blockade of CB1 signaling (with the specific CB1 antagonist N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide [AM251] or CB1 gene knockdown) enhanced hMMC degranulation and increased total numbers without affecting their proliferation in situ. This suggests that inhibiting CB1 signaling induces hMMC maturation from resident progenitor cells within human mucosal stroma. hMMC maturation was induced at least in part through upregulating stem cell factor production. Both the prototypic endocannabinoid anandamide and the CB1-selective agonist arachidonyl-2-chloroethylamide effectively counteracted secretagogue-triggered excessive hMMC degranulation. The current serum-free nasal polyp organ culture model allows physiologically and clinically relevant insights into the biology and pharmacologic responses of primary hMMCs in situ. In human airway mucosa hMMC activation and maturation are subject to a potent inhibitory endocannabinoid tone through CB1 stimulation. This invites one to target the endocannabinoid system in human airway mucosa as a novel strategy in the future management of allergic diseases.

Recovery of Thymic Epithelial Cells and T Cell Immune Reconstitution After Cord Blood Transplantation in Adults Depends On Angiogenesis and Neovascularization

Abstract 4175Umbilical cord blood transplantation (UCBT) is increasingly used as alternative source for allogeneic hematopoietic stem cell transplantation. UCBT in adults results in delayed and insufficient immune reconstitution leading to high infection-related morbidity and mortality. A significant component of immune reconstitution after UCBT involves the thymic regeneration, which can be measured by T-cell receptor rearrangement excision circles (TRECs). TREC levels are a strong predictive factor for overall survival in adult recipients of UCBT. HSCT compromises thymopoiesis due to injury of the thymic microenvironment, particularly thymic epithelial cells (TEC). Human UCB is enriched in endothelial precursors that can sustain thymopoiesis in immunodeficient mice transplanted with human thymic grafts, where they engraft and promote neovascularization and wound healing. We hypothesized that thymic neovascularization induced by the UCB-derived endothelial progenitors might be a critical factor involved in the recovery of TEC, which subsequently support T cell development and expansion via production of interelukin-7 (IL-7) and stem cell factor (SCF; a kit ligand). In the present study we examined whether markers of angiogenesis and neovascularization ANG1 and VEGF were associated with TEC function as determined by assessment of IL-7 and SCF levels and with thymic regeneration as determined by TREC values and recovery of T cell subsets. 27 evaluable patients with a median age of 50 years with hematopoietic malignancies were treated with reduced intensity conditioning (Flu/Mel/rATG) followed by infusion with two sequential UCB grafts and GvHD prophylaxis with tacrolimus and sirolimus. Results are based on 27 evaluable patients. Assessments were done prior to transplantation and at 1, 2, 3, 6 and 12 months after UCBT. At various time points after UCBT, serum levels of IL-7 and SCF displayed a strong correlation (p<0.005) with the levels of ANG1 and VEGF indicating that functional recovery of TEC is associated and coincides with angiogenesis and neovascularization. In contrast, serum levels of IL-7 and SCF displayed a strong inverse correlation (p<0.001) with TREC, CD4+, CD8+ and Treg numbers suggesting that uptake of these cytokines by cognate IL-7R and Kit receptors expressed on immature T cell progenitors resulted in differentiation and expansion of these immature T cells. Similarly, IL-7 and SCF levels displayed a strong inverse correlation (p<0.001) with the ability of T cells to differentiate into pathogen-specific effectors as determined by CMV-specific, IFN-γ ELISpot, underlying the potential clinical implications of these findings. Conversely, VEGF and ANG1 levels positively correlated (p<0.001) with CD4+, CD4+CD25+ and CD4+CD45RA+ numbers at various time points after UCBT. Our results are consistent with a model of TEC recovery supported by VEGF and ANG1, leading to production of SCF and IL-7 by TEC, and uptake of these cytokines by T cell progenitors resulting in their differentiation and expansion. Thus, T cell immune reconstitution after UCBT depends on TEC and is associated with indicators of angiogenesis. These sequential steps in the process of thymic regeneration might represent novel therapeutic targets for improvement of immune reconstitution after UCBT. Disclosures:No relevant conflicts of interest to declare.

Identification of Novel Adult Mesenchymal Progenitors That Can Contribute to HSC Niche

AbstractAbstract 3467The microenvironment (niche) cues hematopoietic stem cell (HSC) receives play an important role in the regulation of their decisions between self-renewal and differentiation. However, the cellular constitution of niches remains poorly understood. We identified three adult progenitor populations near the endosteum based on differential expression of cell surface markers, including CD166, CD146 and Sca1. Upon co-transplantation with fetal skeletal progenitors, Sca1+ progenitors can give rise to CD146+ and CD166+ stroma and phenotypically CXCL12 abundant reticular (CAR) cells in marrow. CD146+ and CD166+ progenitors, on the other hand, form bone without marrow cavity. Multiplex single cell qRT-PCR reveals all three progenitors expressed high levels of genes involved in HSC maintenance. In vitro co-culture assay demonstrated that all three progenitors could preserve HSC long-term multi-lineages reconstitution capability. Furthermore, disruption of stem cell factor (SCF) production in Sca1+ progenitor severely limits its ability to support HSC both in culture condition and after transplantation. Our results suggested that Sca1+, CD146+, and CD166+ mesenchymal progenitors and their progeny collaborate to provide supportive environment for hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Lipopolysaccharide-induced Stem Cell Factor Messenger RNA Expression and Production in Odontoblast-like Cells

IntroductionThis study was done to evaluate the mechanism involved in stem cell factor (SCF) expression and production by lipopolysaccharide (LPS)-stimulated odontoblast (OD)-like cells and to investigate the signal transduction pathway activated in the process. MethodsODs-like cells (MDPC-23) were stimulated with different LPS concentrations for 1, 6, and 24 hours. SCF expression in OD-like cells was analyzed by reverse-transcriptase polymerase chain reaction, and SCF production was assessed by enzyme-linked immunosorbent assay. In another set of experiments, OD-like cells were pretreated with dexamethasone (DEX), MK886 (MK), p42/44 inhibitor (PD 98059 [PD]), p38 inhibitor (SB 202190 [SB]), or PI3K inhibitor (wortmannin [Wort]) for 30 minutes followed by stimulation with LPS (0.1 μg/mL) for 1 hour. ResultsOD-like cells stimulated with LPS (0.1 μg/mL) for 1 hour expressed SCF, but SCF production decreased with increasing LPS concentrations (1, 10, and 100 μg/mL). DEX and MK were able to inhibit SCF messenger RNA (mRNA) expression. PD, SB, and Wort inhibited the SCF mRNA expression. ConclusionsLPS-induced SCF mRNA expression and production in OD-like cells occur via leukotriene production or cytokine and/or chemokine formation, activating the p42/44, p38, and PI3K pathways. Data suggest that SCF released by OD-like cells may act as immune response modulators.

Proteinase-activated receptors in the endometrium and endometriosis

Proteinase-activated receptors (PARs) are G protein-coupled receptors activated by various proteinases. PARs play important roles in haemostasis, thrombosis, and inflammation. PAR₁ and PAR₂ are expressed in endometrial cells from the eutopic endometrium and endometriotic cells derived from endometriotic lesions. A typical activator of PAR₁, thrombin, and a typical activator of PAR₂, tryptase, are produced in the endometrium as well as endometriotic lesions. PAR₁ activation in endometrial stromal cells induces production of vascular endothelial growth factor and matrix metalloproteinases, and increases activities of tissue-type and urokinase-type plasminogen activator. PAR₂ activation in endometrial stromal cells stimulates interleukin (IL)-8 and stem cell factor production and proliferation of the cells. PAR₁ activation in endometriotic stromal cells induces production of IL-8, monocyte chemotactic protein-1, and cyclooxygenase-2, and proliferation of the cells. PAR₂ activation in endometriotic stromal cells increases secretion of IL-6 and IL-8, and the number of the cells. These findings indicate a wide range of function of PAR₁ and PAR₂ in the endometrium and endometriosis, and suggest PAR₁ and PAR₂ as possible therapeutic targets for endometriosis.

Proteinase-activated receptors in the endometrium and endometriosis

Proteinase-activated receptors (PARs) are G protein-coupled receptors activated by various proteinases. PARs play important roles in haemostasis, thrombosis, and inflammation. PAR₁ and PAR₂ are expressed in endometrial cells from the eutopic endometrium and endometriotic cells derived from endometriotic lesions. A typical activator of PAR₁, thrombin, and a typical activator of PAR₂, tryptase, are produced in the endometrium as well as endometriotic lesions. PAR₁ activation in endometrial stromal cells induces production of vascular endothelial growth factor and matrix metalloproteinases, and increases activities of tissue-type and urokinase-type plasminogen activator. PAR₂ activation in endometrial stromal cells stimulates interleukin (IL)-8 and stem cell factor production and proliferation of the cells. PAR₁ activation in endometriotic stromal cells induces production of IL-8, monocyte chemotactic protein-1, and cyclooxygenase-2, and proliferation of the cells. PAR₂ activation in endometriotic stromal cells increases secretion of IL-6 and IL-8, and the number of the cells. These findings indicate a wide range of function of PAR₁ and PAR₂ in the endometrium and endometriosis, and suggest PAR₁ and PAR₂ as possible therapeutic targets for endometriosis.

Proteinase-activated receptors in the endometrium and endometriosis

Proteinase-activated receptors (PARs) are G protein-coupled receptors activated by various proteinases. PARs play important roles in haemostasis, thrombosis, and inflammation. PAR1 and PAR2 are expressed in endometrial cells from the eutopic endometrium and endometriotic cells derived from endometriotic lesions. A typical activator of PAR1, thrombin, and a typical activator of PAR2, tryptase, are produced in the endometrium as well as endometriotic lesions. PAR1 activation in endometrial stromal cells induces production of vascular endothelial growth factor and matrix metalloproteinases, and increases activities of tissue-type and urokinase-type plasminogen activator. PAR2 activation in endometrial stromal cells stimulates interleukin (IL)-8 and stem cell factor production and proliferation of the cells. PAR1 activation in endometriotic stromal cells induces production of IL-8, monocyte chemotactic protein-1, and cyclooxygenase-2, and proliferation of the cells. PAR2 activation in endometriotic stromal cells increases secretion of IL-6 and IL-8, and the number of the cells. These findings indicate a wide range of function of PAR1 and PAR2 in the endometrium and endometriosis, and suggest PAR1 and PAR2 as possible therapeutic targets for endometriosis.

Immune Reconstitution After Cord Blood Transplantation in Adults Depends on Activity of Thymic Epithelial Cells and Vascular Endothelial Elements,

Abstract 4075Umbilical cord blood (UCB) grafts are increasingly used as sources of hematopoietic stem cells. UCBT in adults results in delayed and insufficient immune reconstitution leading to high infection-related morbidity and mortality. We recently determined that thymic regeneration as identified by T-cell receptor rearrangement excision circles (TREC) is a strong predictive factor for clearance of CMV viremia and for overall survival in adult UCBT recipients. We were able to detect TRECs by 100 days that was faster than in other adult UCBT studies reported previously, in which TRECs remained undetectable during the first year. A significant difference between our study and previous reports was the use of 2 UCB units in all our patients whereas only a single unit was used in the previous trials. Human UCB is enriched in endothelial precursors that sustain thymopoiesis in SCID mice transplanted with human thymic grafts where they engraft and promote neovascularization and wound healing. The loss of thymopoietic capacity after allogeneic stem cell transplantation results from injury of the thymic microenvironment, particularly of thymic epithelial cells (TEC). We hypothesized that thymic neovascularization induced by UCB-derived endothelial progenitors might be a critical factor promoting early recovery of TEC, which support T cell development and expansion via production of interelukin-7 (IL-7) and stem cell factor (SCF; a kit ligand). In the present study we examined whether thymic regeneration and immune reconstitution in adult recipients of double UCBT was associated with IL-7 and SCF levels and with activity of endothelial progenitors. Thirty-two patients with a median age of 50 years with hematopoietic malignancies were treated with reduced intensity conditioning (Flu/Mel/rATG) followed by infusion with two sequential UCB units. GvHD prophylaxis was with tacrolimus and sirolimus. Results are based on 27 evaluable patients. Assessments were done prior to and at 1, 2, 3, 6 and 12 months after UCBT. Serum levels of IL-7 displayed a 3-fold increase at 1 month after UCBT and gradually declined to the pre-transplant levels at 12 months. SCF peaked at 2 months after UCBT and gradually decreased to the pre-transplant levels by 12 months. Strikingly, IL-7 and SCF levels displayed a strong inverse correlation (p<0.001) with TREC, CD4, CD8 and Treg cell numbers at various time intervals after UCBT suggesting that uptake of these cytokines by cognate IL-7R and Kit receptors expressed on the surface of immature T cell progenitors resulted in differentiation and expansion of these T cell populations. Moreover, there was a strong inverse correlation (p<0.001) of IL-7 and SCF levels with the ability of T cells to differentiate into pathogen-specific effectors as determined by CMV-specific, IFN-γ ELISpot, underlying the potential clinical implications of these findings. To determine whether TEC activity correlated with endothelial lineage engraftment and neovascularization, we assessed levels of serum thrombomodulin (TM), which is expressed by metabolically active vascular endothelial cells. Serum TM at 1, 2, 3 and 6 months strongly correlated (p<0.005) with the levels of IL-7 and SCF at various time points after UCBT. Thus, quantitative and qualitative T cell immune reconstitution after UCBT depends on IL-7 and SCF and is associated with activity of vascular elements. These results support the intriguing hypothesis that infusion of 2 UCB units might have a cell dosage effect on thymic regeneration and immune reconstitution similar to that seen with improved rates of hematologic engraftment in double versus single UCB recipients. Disclosures:No relevant conflicts of interest to declare.