Hemopoietic Stem Progenitor Cells Research Articles

MTORC1-selective activation of translation elongation promotes disease progression in chronic lymphocytic leukemia

Targeted deletion of Raptor, a component of mechanistic target of rapamycin complex 1 (mTORC1), reveals an essential role for mTORC1 in initiation/maintenance of leukemia in a CLL model, resulting from a failure for haemopoietic stem/progenitor cells (HSPCs) to commit to the B cell lineage. Induction of Raptor-deficiency in NSG mice transplanted with Mx1-Raptor CLL progenitor cells (PKCα-KR-transduced HSPCs) after disease establishment revealed a reduction in CLL-like disease load and a significant increase in survival in the mice. Interestingly in an aggressive CLL-like disease model, rapamycin treatment reduced disease burden more effectively than AZD2014 (dual mTORC1/2 inhibitor), indicating a skew towards mTORC1 sensitivity with more aggressive disease. Rapamycin, but not ibrutinib, efficiently targeted the eEF2/eEF2K translation elongation regulatory axis, downstream of mTORC1, resulting in eEF2 inactivation through induction of eEF2T56 phosphorylation. mTOR inhibitor treatment of primary patient CLL cells halted proliferation, at least in part through modulation of eEF2K/eEF2 phosphorylation and expression, reduced protein synthesis and inhibited expression of MCL1, Cyclin A and Cyclin D2. Our studies highlight the importance of translation elongation as a driver of disease progression and identify inactivation of eEF2 activity as a novel therapeutic target for blocking CLL progression.

CD123 and CD33 Co-Targeting By Balanced Signaling on CAR-CIK Cells Reduces Potential Off-Target Toxicity While Preserving the Anti-Leukemic Activity of Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients. Adoptive cell therapy by chimeric antigen receptor (CAR) T cells demonstrated a high therapeutic potential in B-acute lymphoblastic leukemia, but translation in AML is limited by the absence of an ideal tumor-specific antigen. CD123 (known as IL-3 receptor alfa) and CD33 satisfy several features of ideal target antigens as they are expressed in almost all AML patients (mainly overexpressed in NPM1 and FLT-3 mutated AML), and conserved at disease relapse . Despite that, CD33 expression on hemopoietic stem/progenitor cells (HSPC) induced prolonged myelotoxicity after anti-CD33 CAR-T cell therapy and CD123 expression on endothelium was responsible for severe capillary leak syndrome after anti-CD123 CAR therapy. With the aim to improve selectivity for CD123+/CD33+ AML cells while minimizing toxicity against healthy cells, we probe a dual targeting model by Cytokine Induced Killer (CIK) cells co-expressing a first generation low affinity anti-CD123 IL-3 zetakine (IL3z.CAR) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR). This trans-signaling strategy could allow: 1) low toxicity profile against CD123+ endothelial cells and HSPC, due to a reduced cell activation given by the suboptimal first-generation CAR signal; 2) no or low myelotoxicity against CD33+ HSPC cells, due to absence of CIK cell activation upon the sole costimulatory signal engagement; 3) full CAR-CIK activation only against double expressing CD123 + / CD33 + leukemic cells.Fresh and frozen peripheral blood mononuclear cells were transduced with retroviral vectors during the CIK cell differentiation process . The functional activity of CAR-CIK cells was assessed in vitro by means of short- and long- term cytotoxicity and cytokine production assays upon challenge with different CD123/CD33 positive AML cell lines (THP-1 and KG-1) or with a CD123 positive human endothelial cell line (TIME). To assess the myelotoxicity against HSPC, CD34+ cells were sorted after 24h co-culture with CAR-CIK cells and mixed with methylcellulose-based semisolid medium to evaluate Colony Forming Units (CFU) after 14 days through an automated imaging and counting system for hematopoietic colonies (Stemvision). The in vivo anti-leukemic activity was evaluated by monitoring luciferase-expressing KG1 AML cell line growth over time in NSG mice treated with 3 infusions of all different CAR-CIK conditions.Dual CAR-CIK cells (IL-3z.CAR/CD33.CCR) display a potent and specific in vitro anti-leukemic efficacy against all the AML cell lines tested, compared to single targeting IL-3z.CAR and non-transduced CIK cells. To further minimize toxicities against healthy cells exploiting differences in CD123 antigen density between leukemic and healthy cells, we generated a low affinity dual CAR decreasing the IL3z.CAR binding affinity to CD123, by site-directed mutagenesis. Low affinity dual CAR-CIK cells show irrelevant cytotoxicity against the TIME endothelial cell line, comparable to non-transduced CIK cells, while preserving the efficacy against THP1 and KG1 AML cell lines. As a confirm of the reduced reactivity against endothelial cells, low affinity dual CAR-CIK cells produce the same levels of IL-2 and IFNγ either alone or in the presence of TIME cell line. Low affinity dual CAR-CIK cells also preserve the in vitro CFU-E, CFU-GM and CFU-GEMM colony forming capacity as compared to wild type (wt) dual CAR-CIK and wt IL-3z.CAR-CIK cells, highlighting the possibility to find a therapeutic window to minimize toxicity on healthy cells. The anti-leukemic activity of low affinity dual CAR-CIK cells has been confirmed also in vivo, with a significant suppression of leukemic growth against KG1 AML cell line. Mice treated with 3 infusions of low affinity dual CAR-CIK cells exhibited a significant better anti-leukemic profile, when compared to IL-3-single targeting CAR-CIK cells in terms of tumor growth and overall survival.These preclinical data demonstrate a powerful antitumor efficacy mediated by low affinity dual targeting IL-3z.CAR/CD33.CCR CIK cells against AML targets without any relevant in vitro toxicity on HSPC and endothelial cells, offering a proof-of-concept strategy to increase selectivity for CD123+/CD33+ AML cells whilst reducing the risk of “on-target off-tumor toxicity”. DisclosuresPerriello: Novartis: Other: Advisory Board. Martelli: Abbvie, Amgen, Celgene, Janssen, Novartis, Pfizer, Jazz Pharmaceuticals: Honoraria. Falini: Rasna Therapeutics: Honoraria. Biondi: Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria.

Distinct profile of CD34+ cells and plasma-derived extracellular vesicles from triple-negative patients with Myelofibrosis reveals potential markers of aggressive disease

BackgroundMyelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10–15% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TN patients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis.MethodsPeripheral blood was collected from MF patients (n = 29; JAK2V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34+ cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34+ cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, IL6) or after co-culture with EVs from MF patients/HD.ResultsThe absolute numbers of circulating CD34+ cells were massively increased in TN patients. We found that TN CD34+ cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TN patients. Compared to JAK2V617F-mutated patients, the GEP of TN CD34+ cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2V617F-mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TN CD34+ cells. Along with a specific microRNA signature, the circulating EVs from TN patients are enriched with miR-361-5p.ConclusionsDistinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF.

Prognostic value of soluble angiotensin II receptor 1 and soluble angiotensin converting enzyme (CD 143) in patients with acute leukemia

AbstractBackgroundThe renin-angiotensin system (RAS) is a bioenzymic cascade that plays an integral role in cardiovascular homoeostasis by influencing vascular tone, fluid and electrolyte balance and the sympathetic nervous system. RAS was viewed as a circulating endocrine system, whereby renin released from the juxtaglomerular cells of the kidney cleaves the liver-derived macroglobulin precursor angio-tensinogen, to produce the inactive decapeptide angiotensin I, which is then converted to the active octapeptide Ang II by angiotensin converting enzyme (ACE) within the pulmonary. There is increasing evidence that Ang II, a major regulator of blood pressure and cardiovascular homeostasis, is involved in the regulation of cell proliferation, angiogenesis, inflammation and tissue remodeling, which suggests that this peptide might also play a role in cancer. Ang II is the main effector of the RAS and it alternatively binds to either Ang II T1R or Ang II T2R. The Ang II T1R and Ang II T2R can act as antagonists, and mediate effects on cell migration and proliferation of metastatic cancer cells and hemopoietic stem-progenitor cells. Components of the RAS are frequently differentially expressed in various cancers in comparison with their corresponding non- malignant tissue. Yet, the RAS has not been fully elucidated in patients with acute leukemia. Objective: The aim of the present work was to study serum level of Angiotensin II receptor type 1 and the soluble angiotensin converting enzyme (CD143) in patients with acute leukemia in order to extrapolate their possible prognostic value.Subjects and MethodsThe present study included 20 healthy volunteers clinically free from hypertension or sarcoidosis, 20 patients of newly diagnosed AML and 20 patients of newly diagnosed ALL. Blood samples were collected from all subjects and the level of serum ACE and serum Ang IIT1R were measured by enzyme linked immunossorbent assay.ResultsThe activity of ACE (U/L) and the concentration of Ang IIT1R (U/L) in patients groups with either AML or ALL before therapy were significantly higher than in control group. After therapy, the activity of the enzyme and its receptor concentration in both groups of patients were significantly decreased but still significantly higher than in normal control subjects.ConclusionsEstimating the serum level of ACE and soluble Ang IIT1R is of informative diagnostic and prognostic value. Estimation serum level of ACE and Ang IIT1R levels in patients with either AML or ALL is of value in deciding the treatment protocol.

The Malignant Hemopoietic Clone of Triple Negative Patients with Myelofibrosis Shows in Vitro Functional Defects but Is Highly Responsive to the Pro-Survival Signals of Circulating Autologous Microvesicles

BackgroundMyelofibrosis (MF) is a clonal hemopoietic stem/progenitor cells (HSPCs) disorder with mutations in 3 driver genes (JAK2, MPL, or CALR) and inflammatory microenvironment. Triple negative (TN) patients (pts) do not carry the 3 driver mutations and display a significantly worse survival-rate which can be partially due to a greater mutation complexity. Circulating microvesicles (MVs; 0.1-1 μm), most of megakaryocytes (MKMVs) and platelets (PMVs) origin, play a role in intercellular signalling and are increased in inflammation and cancer including MF. We hypothesized that in TN pts the HSPCs and/or the inflammatory microenvironment show a more aggressive and malignant phenotype.AimsTo identify a biomolecular signature of TN pts by comparatively evaluating the circulating HSPCs behaviour and their functional interplay with the inflammatory microenvironment.MethodsEDTA-anticoagulated peripheral blood (PB) was collected from 16 MF pts (WHO-2016 criteria-JAK2(V617F) mutated (n=10) and TN (n=6)) and 20 age/sex-matched healthy donors (HD). Driver mutation status was obtained by PCR/NGS. Plasma MVs were isolated by ultracentrifugation (45.000 rpm for 2 hours), counted by Nanosight technology and phenotypically characterized for PMVs (CD61+CD62P+) and MKMVs (CD61+CD62P-) by flow cytometry (size 0.5-0.9 µm). in vitro survival (24 hours), CXCL12-driven migration (12 hours) and clonogenic ability of immunomagnetically isolated CD34+ cells from pts PB or cord blood (CB; n=10) was performed in the presence/absence of the isolated MVs or IL-1β, TNF-α and IL-6 (alone/ in combination). Incorporation of CFSE-labelled MVs into CD34+ cells was analyzed by confocal microscopy. MicroRNA (miRs) profile analysis of the isolated MVs and Gene expression profile (GEP) analysis of CD34+ cells from 3 JAK2(V617F) mutated, 3 TN pts and 3 HD/CB was performed. Plasma inflammatory cytokines levels were evaluated by ELISA.ResultsWe firstly compared the TN and JAK2(V617F) CD34+ cells. TN CD34+ cells showed decreased in vitro survival and clonogenic ability but comparable migration capacity (Fig. 1 A, B, C). Consistently, comparing the GEP, the expression of selected anti-apoptotic (TSPYL5, GFI-1 and FCMR) and pro-apototic (TNFSF10, TP53INP1) genes was significantly down- and up-regulated, respectively, in TN CD34+ cells. We then investigated whether signals from the microenvironment may affect the in vitro behaviour of the TN CD34+ cells. IL-1β, IL-6 and TNF-α plasma levels were significantly increased (p<0.05) in MF pts. However, at variance with the JAK2(V617F) mutated counterparts, survival and clonogenic ability of TN CD34+ cells were not significantly promoted by IL-1β, IL-6 and TNF-α, alone or in combination. This was due, at least in part, to the reduced expression of IL-6 and TNF-α (type I) receptors on TN CD34+ cells. We then analyzed the phenotype of the isolated circulating MVs showing that the PMVs were significantly increased (p<0.05) in the JAK2(V617F) mutated pts only. Conversely, the MKMVs were significantly decreased in both pts groups (p<0.01) as compared to the HD counterparts (Fig. 1 D, E). When we co-cultured CD34+ cells with the isolated MVs we found that TN MVs significantly increased the survival of TN CD34+ cells but inhibited CB CD34+ cells survival (p<0.05, respectively). Conversely, JAK2(V617F) MVs did not significantly affected the survival of CB/pts CD34+ cells. HD MVs significantly increased the survival of CB CD34+ cells (p<0.05) only (Fig. 1 F, G, H). Of note, MVs from pts were transferred into cytosol after co-cultured with CB CD34+ cells (Fig. 1 I). Consistently, comparing the miRs profile of pts and HD MVs, various pro-apoptotic (+) and anti-apoptotic (-) miRs were upregulated. Specifically, miR-155(-), 24(-) and 222(-) were upregulated in TN MVs only; miR-21(-), 19a(-), 34a-5p(+), 423-5p(+) were upregulated in both JAK2(V617F) and TN MVs. Further, miRs regulating inflammation (miR-146a) and proliferation (miR-1274A, downregulated in TN vs JAK2(V617F) MVs), were overexpressed in pts MVs.ConclusionsOur results demonstrate that TN CD34+ cells show in vitro defective function and are unresponsive to the inflammatory microenvironment. Interestingly, only the autologous circulating MVs promoted the malignant clone, suggesting that MVs, as signals from microenvironment, may have a pathogenetic role in TN MF and may be the target of novel therapeutic approaches. [Display omitted] DisclosuresFabbri:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Palandri:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Mobilized Peripheral Blood versus Cord Blood: Insight into the Distinct Role of Proinflammatory Cytokines on Survival, Clonogenic Ability, and Migration of CD34+Cells

Inflammation may play a role in cancer. However, the contribution of cytokine-mediated crosstalk between normal hemopoietic stem/progenitor cells (HSPCs) and their (inflammatory) microenvironment is largely elusive. Here we compared survival, phenotype, and function of neonatal (umbilical cord blood (CB)) and adult (normal G-CSF-mobilized peripheral blood (mPB)) CD34+ cells after in vitro exposure to combined crucial inflammatory factors such as interleukin- (IL-) 1β, IL-6, tumor necrosis factor- (TNF-) α, or tissue inhibitor of metalloproteinases-1 (TIMP-1). To mimic bone marrow (BM) niche, coculture experiments with normal BM stromal cells (BMSCs) were also performed. We found that combined inflammatory cytokines increased only the in vitro survival of CB-derived CD34+ cells by reducing apoptosis. Conversely, selected combinations of inflammatory cytokines (IL-1β + TNF-α, IL-6 + TNF-α, and IL-1β + TNF-α + TIMP-1) mainly enhanced the in vitro CXCR4-driven migration of mPB-derived CD34+ cells. TNF-α, alone or in combination, upregulated CD44 and CD13 expression in both sources. Finally, BMSCs alone increased survival/migration of CB- and mPB-derived CD34+ cells at the same extent of the combined inflammatory cytokines; importantly, their copresence did not show additive/synergistic effect. Taken together, these data indicate that combined proinflammatory stimuli promote distinct in vitro functional activation of neonatal or adult normal HSPCs.

Abstract 4077: Crucial factors of the inflammatory microenvironment (IL-1β/TNF-α/TIMP-1) promote the selection of highly malignant hemopoietic clone of myelofibrosis

Abstract Myelofibrosis (MF) is a clonal disorder of the hemopoietic stem/progenitor cells (HSPCs). Together with molecular abnormalities (mutations in JAK2, Calreticulin (CALR) and MPL genes), chronic inflammation is the major hallmark of MF pathogenesis. It is argued that the up-regulated production of pro-inflammatory cytokines selects for the malignant clone. However, the key players linking inflammation and cancer in MF are still poorly known. Here, we investigated the in vitro effects of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Tissue Inhibitor of Metalloproteinases (TIMP)-1 and ATP) on the functional behaviour of MF-derived HSPCs. Plasma TIMP-1, TNF-α and IL-1β were measured by ELISA assay. Circulating CD34+ cells from MF patients (JAK2V617F (10 cases) and CALR (10 cases) mutations) or cord blood (CB; 8 samples) were functionally characterized after incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), cell cycle and clonogenic capacity. Migration was assessed towards a C-X-C motif chemokine 12 (CXCL12) gradient in the presence or absence of the pro-inflammatory factors. MF-derived CD34+ cells were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence of the pro-inflammatory factors and then evaluated for survival. Regardless mutation status, the plasma levels of IL-1β, TNF-α and TIMP-1 are increased in MF patients and the presence of pro-inflammatory cytokines confers a survival advantage of MF-derived CD34+/CD34+CD38- cells. In addition, MF-derived CD34+ cells promoted cell-cycle progression to the S-phase. Whereas in the JAK2V617F mutated group, the addition of IL-1β or TNF-α ± TIMP-1 impaired the erythroid clonogenic output of the CALR mutated patients. Of note, IL-1β + TNF-α ± TIMP-1 highly promoted the in vitro migration of MF-derived CD34+ cells in the presence of CXCL12. Intriguingly, MF-derived CD34+ cells revealed increased clonogenic ability after migration toward CXCL12 and IL-1β + TNF-α ± TIMP-1, suggesting a pivotal role of inflammation in the selection of the malignant clone. Finally, co-cultures of normal MSCs with MF-derived CD34+ cells sustained the survival effects of IL-1β+TNF-α±TIMP-1. Overall, our data indicate that the behavior of the MF-derived HSPCs can be influenced by regulatory signals provided by the microenvironment through the cooperation between various pro-inflammatory factors. The interplay of these pro-inflammatory factors promotes and selects the circulating MF-derived HSPCs with higher proliferative activity, clonogenic potential and migration ability. Targeting these micro-environmental interactions may be a clinically relevant approach. Citation Format: Daria Sollazzo, Dorian Forte, Nicola Polverelli, Marco Romano, Margherita Perricone, Sofia Fatica, Francesco Lapi, Emanuela Ottaviani, Martinelli Giovanni, Nicola Vianelli, Michele Cavo, Maria Pantaleo, Francesca Palandri, Lucia Catani. Crucial factors of the inflammatory microenvironment (IL-1β/TNF-α/TIMP-1) promote the selection of highly malignant hemopoietic clone of myelofibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4077.

Protective Effects of Tannins in Sanguisorbae Radix on Myelosuppression Mice

ObjectiveTo explore the protective effects of tannins in Sanguisorba Radix (TSR) on myelosuppression mice induced by cyclophosphamide (CTX). MethodsTSR was ig given at the dose of 20 mg/kg for 10 d after ip administration of CTX (200 mg/kg). ResultsTSR could significantly increase the numbers of white blood cells, red blood cells, and platelets of myelosuppression in mice. And it could accelerate bone marrow haemopoietic stem/progenitor cells (HSPCs) in myelosuppression mice and enhance cell proliferation by promoting cell cycles from G0/G1 phase to access into S and G2/M phases, then the reduced number of HSPCs induced by CTX was reversed. Moreover, TSR could increase the mRNA and protein expression levels of O(6)-methylguanine-DNA methyltransferase (MGMT) in HSPCs of myelosuppression mice. ConclsionTSR has a protective function against CTX-induced myelosuppression. The mechanism might be related to protecting hematopoietic stem cells of bone marrow, stimulating hematopoiesis recovery, as well as preventing the apoptosis of hematopoietic stem cells induced by CTX.

ZNF16 (HZF1) promotes erythropoiesis and megakaryocytopoiesis via regulation of the c-KIT gene

We previously characterized the zinc finger protein gene HZF1 [also known as ZNF16 (zinc finger protein 16)] and demonstrated its important roles in erythroid and megakaryocytic differentiation of K562 cells. In the present study, we investigated its effect on erythroid and megakaryocytic differentiation of HSPCs (haemopoietic stem/progenitor cells). We observed up-regulation of ZNF16 during erythroid and megakaryocytic differentiation of the CD34+ HSPCs, and demonstrated that ZNF16 promotes erythroid and megakaryocytic differentiation by gain-of-function and loss-of-function experiments. Using a luciferase reporter and ChIP assays ZNF16 was demonstrated to bind to the c-KIT gene promoter and inhibit its expression in K562 cells. Enforced expression and knockdown of ZNF16 down-regulated and up-regulated the expression of the c-KIT gene in K562 cells and HSPCs respectively. Significantly decreased levels of the c-Kit protein were observed following erythroid and megakaryocytic differentiation of K562 and CD34+ cells. The knockdown of c-KIT partially rescued the differentiation inhibition caused by ZNF16 knockdown. The knockdown of c-KIT also blocked the activity of the c-Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK/c-Jun signal pathway and reduced further the level of HEY1 (hes-related family bHLH transcription factor with YRPW motif 1), a repressor of GATA1 (GATA-binding protein 1) transcription, which finally up-regulated the expression of GATA1, a central regulator of erythroid and megakaryocytic differentiation. In conclusion the results of the present study demonstrate that ZNF16 plays an important role in erythropoiesis and megakaryocytopoiesis via its regulation of the c-Kit/c-Raf/MEK/ERK/c-Jun/HEY1/GATA1 cascade.

Maternal plasma or human serum albumin in wash buffer enhances enrichment and ex vivo expansion of human umbilical cord blood CD34+ cells

Umbilical cord blood is a valuable source of haemopoietic stem/progenitor cells (HSC) for transplantation. This study explored the effect of maternal plasma/human serum albumin (HSA) in the purification and culture conditions of CD34+ cells derived from human umbilical cord blood. During CD34+ cell enrichment, including maternal plasma or HSA instead of fetal bovine serum (FBS) in the wash buffer, significantly increased the purity and the fold expansion of CD34+ cells. The increase in fold expansion of CD34+ cells was independent of CD34+ cell purity before expansion. With FBS, the mean fold expansion of CD34+ cells and total nucleated cells on day 7 was 9.7 +/- 5.5 and 39.7 +/- 13.7 respectively. The use of maternal plasma increased the mean fold expansion of CD34+ cells and total nucleated cells on day 7 to 28.2 +/- 6.7 and 71.5 +/- 15.4 respectively. When HSA was added to wash buffer, the mean fold expansion of CD34+ cells and total nucleated cells were 30.4 +/- 10.5 and 83.5 +/- 24.8 respectively. No statistical significance was found between using HSA and maternal plasma on total cell and CD34+ cell expansion. We propose that HSA in maternal plasma was responsible for the positive effect on CD34+ cell enrichment and expansion.

Haemopoietic and neuroglial progenitors are promoted during cord blood ex vivo expansion.

Umbilical cord blood (UCB) has emerged as a promising source of haemopoietic stem ⁄ progenitor cells (HSPC) for human bone marrow reconstitution, the limitation being the low harvestable number of HSPC from a single UCB sample. In developing our UCB ex vivo expansion strategies, our group designed negative immunomagnetic selection systems to deplete developing haemopoietic cells from HSPC. The resulting highly purified non-committed lineage negative (Lin) cell population represents 0AE12% (± 0AE02 SEM, n 1⁄4 9) of original UCB mononuclear cells. In liquid culture (Iscove’s modified Dulbecco’s medium – 10% fetal calf serum, supplemented with thrombopoietin, Flt-3 ligand and c-Kit-ligand), Lin cells proliferated and maintained haemopoietic colonies for at least 5 weeks. In three independent experiments, Lin cells also produced a distinct adherent cell population showing neuroglial cell morphology. Further analysis with laser-scanning confocal microscopy revealed (i) active cell–cell communication through extensive membrane projections from these adherent cells (top image) and (ii) positivity for Glial Fibrillary Acidic Protein, a neuroglial marker (immuno-fluorescence bottom image). These results support the existence of a cell population with multi-tissue differentiation ability within UCB. Our findings widen the potential of UCB as an accessible and ethically sound stem cell source for treatment of haematological diseases, and possibly for certain neurological disorders.

Stromal support augments extended long-term ex vivo expansion of hemopoietic progenitor cells.

Current technology to numerically expand hemopoietic stem/progenitor cells (HSPC) ex vivo within 1 to 2 weeks is insufficient to warrant significant gain in reconstitution time following their transplantation. In order to more stringently test the parameters affecting HSPC expansion, we followed ex vivo cultures of CD34+-selected umbilical cord blood (UCB) HSPC for up to 10 weeks and investigated the effects of stromal support and cytokine addition. The cytokine combinations included FL + TPO, FL + TPO plus SCF and/or IL6, or SCF + IL6. To identify the HSPC in uncultured and cultured material, we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ subsets by phenotyping. The highest fold-increase obtained for CD34+ and CD34+ CD38- cell numbers was, respectively, 1197 and 30,937 for stroma-free and 4066 and 117,235 for stroma-supported cultures. In general, CFC generation increased weekly in FL + TPO containing groups up to week 5 with a 28- to 195-fold expansion whereafter the weekly CFC output stabilized. Stroma support enhanced the expansion of CAFC week 6 maximally 11-fold to 89-fold with FL + TPO + IL6. Cultures stimulated with at least FL + TPO gave an estimated 10- to 14-fold expansion of the ability of CD34+ UCB cells to multilineage engraft the BM of sublethally irradiated NOD/SCID mice at 2 weeks of stroma-free and stroma-supported cultures, while at week 5 and later the estimated SRA decreased to low or undetectable levels in all groups. Our results show that stroma and FL + TPO but also inclusion of bovine serum albumin, greatly increase the long-term generation of HSPC as measured by in vitro assays and is indispensable for long-term expansion of CD34+ CD38- CXCR4+ cells. However, the different surrogate methods to quantify the HSPC (CD34+ CD38-, CFC, CAFC week 6 and SRA) show increasing incongruency with increasing culture time, while especially the phenotypic analysis and the CFC generation greatly overestimate the CAFC and SRA expansion in 10-week cultures.

Increase in circulating SDF-1 after treatment with sulfated glycans. The role of SDF-1 in mobilization.

SDF-1 is a potent chemoattractant for mature white blood cells and hemopoietic stem/progenitor cells (HPCs). An important role for this chemokine in mobilization has been postulated, but in vivo studies directly addressing its effects are lacking. After one injection of fucan sulfate (FucS) or dextran sulfate, plasma levels of SDF-1 are greatly increased in mice or primates. Increases are dose-dependent and correlate with mobilization of HPCs. Elevated levels of circulating SDF-1 appear to be uniquely associated with this treatment, as it was not seen with cytokine or anti-integrin antibody treatments that induce mobilization. In vitro, these sulfated glycans specifically bind to SDF-1 and inhibit SDF-1/heparin binding, suggesting a mechanism of release from sequestration on heparan sulfate proteoglycans in vivo. Although other chemokines including IL8 and cytokines like G-CSF also increase, evidence in GCSFR-deficient mice suggests that at least these two factors are unlikely participants in FucS-induced mobilization. Likewise, although the activity of the metallo-protease MMP9 increases after FucS treatment, experiments in MMP9-/- mice indicate its presence is dispensable for mobilization or SDF-1 release. However, effects of other proteases cannot be ruled out by these experiments. Finally, anti-SDF-1 antibodies partially inhibit FucS-induced mobilization, supporting a causative relationship. Our data offer a unique insight into the mechanism of sulfated glycan-induced mobilization and suggest a novel way of disturbing SDF-1 gradients between bone marrow and peripheral blood.

Biology of the hemopoietic microenvironment.

In adult mammals, hemopoiesis takes place primarily in the bone marrow. The steady-state production of blood cells depends to a large extent on the interaction between hemopoietic stem/progenitor cells (HPC) and the different components of the microenvironment present in the medullary cavity. During the last three decades, in vivo and in vitro studies have allowed significant advances in understanding of the biology of such a hemopoietic microenvironment. Although not evident in histological sections, it is well known that the hemopoietic microenvironment is a highly organized structure that regulates the location and physiology of HPC. The hemopoietic microenvironment is composed of stromal cells (fibroblasts, macrophages, endothelial cells, adipocytes), accessory cells (T lymphocytes, monocytes), and their products (extracellular matrix and cytokines). Microenvironmental cells can regulate hemopoiesis by interacting directly (cell-to-cell contact) with HPC and/or by secreting regulatory molecules that influence, in a positive or negative manner, HPC growth. Recent in vitro studies suggest that functional abnormalities of the hemopoietic microenvironment may be implicated in the manifestation of certain hematological disorders such as aplastic anemia, and acute and chronic myelogenous leukemia. Thus, the characterization of the structure and function of the human hemopoietic microenvironment may have relevance in understanding and treating different hematological disorders.