CD34 Progenitor Cells Research Articles

Colony-forming endothelial cells – candidate culture for tissue vascular engineering: the gene and proteomic profile

Aim. To validate ECFC culture as a candidate culture for vascular tissue engineering using comparative analysis of the proteomic and gene expression profiles in comparison with cultures of human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC).Materials and Methods. ECFC culture was obtained by cultivating peripheral blood mononuclear cells of patients with coronary artery disease. Commercial HCAECs produced by Cell Applications, and HUVECs cultured according to the modified protocol of Jaffe were used as controls.The cells were lysed with TRIzol, and total RNA was isolated using a Purelink RNA Micro Scale Kit with concomitant DNase treatment. Next, rRNA depletion was carried out, followed by the creation of DNA libraries. DNA libraries were quantified using quantitative polymerase chain reaction on a CFX96 Touch Bio-Rad amplifier. DNA libraries were equimolarly mixed and sequenced on HiSeq 2000 (Illumina) with a paired-end reads of 2x125 nucleotides.Conventional western blotting was performed using pan-endothelial markers CD31, vWF, VEG-FR2/KDR, marker of endothelial progenitor cells CD34, markers of epithelial-mesenchymal transition Snail and Slug, and markers of endothelial specification: arterial HEY2, venous COUP-TFII and lymphatic LYVE1, VEGFR2. Dot blotting against 55 angiogenesis-related proteins was performed using Proteome Profiler Human Angiogenesis Array Kit in accordance with the manufacturer's protocol.Results. ECFC overexpresses markers of all three endothelial lineages (KDR, VWF, CD34, NRP2, FLT4 and LYVE1 compared to HCAEC; NOTCH4, DLL2) and LYVE1 compared to HUVEC. Proteomic profiling indicated ECFC as an intermediate population between HCAEC and HU-VEC in term of the expression of HEY2, LYVE1, VEGFR3, Snail and Slug. 261 DEGs were detected between ECFC and HUVEC, and 470 DEGs between ECFC and HCAEC.Conclusion. The gene expression profile of endothelial colony-forming cells corresponds to mature endothelial cells and indicates ECFC as an intermediate population between HCAEC and HUVEC. ECFC culture can be recommended for tissue vascular engineering.

TNFα induces Caspase-3 activity in hematopoietic progenitor cells CD34+, CD33+, and CD41 + of myelodysplastic syndromes

BackgroundCytopenia is the primary feature of Myelodysplastic Syndrome, even in the presence of hypercellular bone marrow. TNFα is recognized as both a proinflammatory, and proapoptotic cytokine with a well established role in promoting apoptosis in MDS. Therefore, TNFα has the potential to be a valuable biomarker for predicting the progression of cytopenia in MDS. This study aims to establish the role of TNFα exposure in triggering apoptosis through caspase-3 activity in CD34+, CD33+, and CD41 + cells in MDS.MethodsThis study is an in vitro comparative experimental research. Bone marrow mononuclear cells were isolated as the source of hematopoietic progenitor cells. Subsequently, CD34+, CD33+, and CD41 + cells were exposed to rhTNFα, and the caspase-3 activity was measured using flowcytometry.ResultsIn MDS CD33 + and CD41 + caspase-3 activity of rhTNFα exposed cells was significantly higher than without exposed cells. The opposite result was found in CD34 + cells, where the caspase-3 activity without rhTNFα exposed cells was significantly higher than rhTNFα exposed cells.ConclusionrhTNFα exposure led to an elevation in caspase-3 activity in MDS progenitor cells, especially in those that had differentiated into myeloid cell CD33 + and megakaryocyte cell CD41+, as opposed to the early progenitor cells CD34+.

Characterize Biomarkers and Mechanisms of Resistance for MDM2 Inhibitors in AML

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease with an aggressive phenotype and poor prognosis. Inactivation of the wild-type p53, through upregulation of the TP53 negative regulator MDM2, is observed in around 50% of AML patients, providing a rationale for targeting MDM2 for the treatment of AML. Small molecule inhibitors of MDM2, including Idasanutlin, have shown efficacy as monotherapy and in combinations. However, patients exhibit heterogeneous responses with frequent drug resistance. We integrated ex vivo drug sensitivity assay, clinical parameters, whole exome sequence (WES), RNAseq, cytokine stimulation, and CRISPR/Cas9 dropout screening data to identify biomarkers that predict idasanutlin response and to elucidate the underlying drug-resistant mechanisms. Surprisingly, besides TP53 itself, we did not find an essential role in losing any of the major TP53 downstream targets alone leading to Idasanutlin resistance from Idasanutlin treated resistant cell lines, CRISPR screening, or individually targeted knockout experiments. Besides TP53 mutations, other major AML mutations, including RAS pathway mutations do not drive Idasanutlin resistance indicated by the patient sample functional screening data and in vitro mutation overexpression models. We observed instead that AML with a high differentiation or mature myeloid signature: including low blast, a high percentage of monocytes/granulocytes/T cells, AML FAB M4/M5 morphology, and a high expression of a myeloid immune signature gene are relatively resistant to Idasanutlin. We further characterized that both healthy/leukemia-associated T cells, granulocytes, and monocytes are intrinsically resistant to idasnutlin due to low expression levels of MDM2 and/or TP53. To examine whether the presence of differentiated cells protects leukemia blast cells from Idasanutlin, we performed a co-culture drug treatment experiment. Strikingly, only leukemia-associated monocytes, but not healthy monocytes, healthy/leukemia T cells or granulocytes, or leukemia blast cells protected leukemia blast cells from apoptosis in the presence or absence of Idasanutlin. Similar results were obtained for the other two MDM2 inhibitors, AMG232 and DS-3032b, and a BCL2 inhibitor, venetoclax. To further uncover which cytokines secreted from the leukemia-associated monocytes confer MDM2 inhibitor resistance, we tested 11 common cytokines, including IL-1a, IL-1b, IL-3, IL-6, IL-8, M-CSF GM-CSF, FLT3, MCP-1, TNFa, and SCF. We observed that IL-1α, IL-1β, IL-3, and GM-CSF markedly induced idasanutlin treatment resistance for AML, but not ALL or CLL patient cells. Interestingly, IL-1α and IL-1β only protect leukemia blasts, but not healthy CD34 hematopoietic stem and progenitor cells from Idasanutlin-induced apoptosis. We further performed RNAseq and immunoblots on IL-1α and IL-1β treated AML, CLL, and ALL cells. We observed the major TP53 downstream pathways remain activated by idasanutlin in the presence of IL-1α and IL-1β. Interestingly, GATA2 is significantly upregulated in IL-1α and IL-1β treated AML, but not CLL or ALL cells in the presence or absence of Idasanutlin. Furthermore, overexpressing GATA2 conferred Idasanutlin resistance at high concentrations. Finally, we observed that combining a FLT3/IRAK dual inhibitor, pacritinib, or an IRAK1 /IL-1 receptor antagonist, anakinra, partially rescued IL-1α and IL-1β mediated drug resistance of MDM2 inhibitors. As such, we uncovered the role of leukemia-associated monocyte in driving intrinsic and extrinsic MDM2 inhibitor resistance and the potential underlying mechanisms.

Efficacy of ticagrelor compared to clopidogrel in improving endothelial function in patients with coronary artery disease: a systematic review.

Ticagrelor and clopidogrel are antiplatelet drugs that act by binding to the adenosine diphosphate P2Y12 receptor. Previous studies have compared between them regarding the endothelial function effect. This systematic review aims to summarize the evidence comparing the efficacy of ticagrelor vs. clopidogrel in improving endothelial function in patients with coronary artery disease (CAD). In August 2021, the Scopus, PubMed, Web of Science, and Cochrane library were searched systematically for eligible trials. We included randomized controlled trials that compared the efficacy of ticagrelor vs. clopidogrel in improving endothelial function in patients with CAD. Seven trials (n = 511) were included in our systematic review. Ticagrelor resulted in a greater elevation of the level of progenitor cells CD34+ KDR+ and CD34+ 133+ (P = 0.036 and P = 0.019, respectively), with a lower rate of endothelial cell apoptosis rate (P < 0.001). Moreover, ticagrelor showed superiority regarding nitric oxide, radical oxygen species, and soluble P-selectin levels (P = 0.03, P = 0.02, and P = 0.019, respectively). Flow-mediated dilation findings differed between the studies (P = 0.004 vs. P = 0.39). Ticagrelor appears to exert an additional improvement in endothelial function compared with clopidogrel in patients with coronary heart disease.

ScPCOR-seq enables co-profiling of chromatin occupancy and RNAs in single cells

Cell-to-cell variation in gene expression is a widespread phenomenon, which may play important roles in cellular differentiation, function, and disease development1–9. Chromatin is implicated in contributing to the cellular heterogeneity in gene expression10–16. Fully understanding the mechanisms of cellular heterogeneity requires simultaneous measurement of RNA and occupancy of histone modifications and transcription factors on chromatin due to their critical roles in transcriptional regulation17,18. We generally term the occupancy of histone modifications and transcription factors as Chromatin occupancy. Here, we report a technique, termed scPCOR-seq (single-cell Profiling of Chromatin Occupancy and RNAs Sequencing), for simultaneously profiling genome-wide chromatin protein binding or histone modification marks and RNA expression in the same cell. We demonstrated that scPCOR-seq can profile either H3K4me3 or RNAPII and RNAs in a mixture of human H1, GM12878 and 293 T cells at a single-cell resolution and either H3K4me3, RNAPII, or RNA profile can correctly separate the cells. Application of scPCOR-seq to the in vitro differentiation of the erythrocyte precursor CD36 cells from human CD34 stem or progenitor cells revealed that H3K4me3 and RNA exhibit distinct properties in clustering cells during differentiation. Overall, our work provides a promising approach to understand the relationships among different omics layers.

Ultrastructural aberrations, histological disruption and upregulation of the VEGF, CD34 and ASMA immunoexpression in the myocardium of anemic albino rats

Iron deficiency anemia (IDA) is a global health problem affecting various body systems and tissues including the cardiovascular system. Several literatures described the associated physiological and clinical changes in the cardiovascular system and heart. However, the associated structural changes were poorly investigated. Therefore, the main aim of the present work was to elucidate whether IDA induces structural changes and alterations in the VEGF, CD34 and ASMA immunoexpression in the myocardium of albino rats. Thirty adult male albino rats were divided into two groups (fifteen rats each); control and anemic. Hematological data for all animals were assessed weekly and statistically analyzed. Three weeks later, animals were sacrificed, and heart specimens were obtained and processed for light and electron microscopy. All hematological parameters showed a statistically significant decrease in the anemic group. Structurally, the anemic group showed markedly degenerated, disrupted and disorganized cardiomyocytes in addition to markedly congested blood vessels, fibroblasts, collagen fibers deposition and perivascular cellular infiltration were noted. Also, positive immunostaining for VEGF, CD34 and ASMA was observed. Ultra-structurally, the myocardium of the anemic group showed disrupted and degenerated myofibrils with degenerated nuclei, perinuclear edema, widened interstitial spaces and marked collagen deposition. Mitochondria markedly increased with abnormal shapes. IDA induced myocardial injury that may propagate to regeneration through activated CD34 progenitor cells and increased VEGF or to degeneration and fibrosis through collagen fibers deposition and enhanced ASMA. So, early diagnosis and treatment of IDA is mandatory to avoid the associated myocardial structural changes.

Cyclin A1 Expression in Leukemia and Normal Hematopoietic Cells

Human cyclin A1 is a newly cloned, tissue-specific cyclin that is prominently expressed in normal testis. In this study, we showed that cyclin A1 was highly expressed in a subset of leukemia samples from patients. The highest frequency of cyclin A1 overexpression was observed in acute myelocytic leukemias, especially those that were at the promyelocyte (M3) and myeloblast (M2) stages of development. Cyclin A1 expression was also detected in normal CD34(+) progenitor cells. The expression of cyclin A1 increased when these cells were stimulated to undergo myeloid differentiation in vitro. Taken together, our observations suggest that cyclin A1 may have a role in hematopoiesis. High levels of cyclin A1 expression are especially associated with certain leukemias blocked at the myeloblast and promyelocyte stages of differentiation.

Endothelial Progenitor Cell CD34+ and CD133+ Concentrations and Soluble HLA-G Concentrations During Pregnancy and in Cord Blood in Undifferentiated Connective Tissue Diseases Compared to Controls.

The objective of this study is to evaluate endothelial progenitor cells (EPCs) CD34+ CD133- and CD34+ CD133+ and soluble HLA-G (sHLA-G) concentrations among undifferentiated connective tissue disease (UCTD) subjects, compared to controls, during pregnancy and in cord blood. This is a case-control study including 29 controls and 29 UCTDs. CD34+ CD133-, CD34+ CD133+, and sHLA-G concentrations were detected in maternal plasma and in cord blood. This study was approved by the Medical-Ethical Committee of our Institution (Current Research Project N. 901-rcr2017i-23 of IRCCS Foundation Policlinico San Matteo of Pavia). Circulating CD34+ CD133- and CD34+ CD133+ counts and sHLA-G (soluble human leucocyte antigen G) concentrations in maternal peripherical blood were higher in UCTD compared to those in controls in first and third trimester of pregnancy and at delivery (p< 0.001). Maternal CD34+ CD133- and CD34+ CD133+ counts were strongly and significantly correlated in UCTD (Spearman's rho = 0.79, p< 0.0001) but not in controls (Spearman's rho = 0.10, p= 0.35). Cord blood CD34+ CD133- and CD34+ CD133+ median counts and median sHLA-G concentrations were higher among UCTD subjects than in controls (p< 0.001). Cord blood CD34+ and CD133+ counts were inversely and significantly correlated with sHLA-G concentrations among UCTDs, but not in controls. Early UCTD is characterized by increased EPC levels in maternal plasma and in cord blood and higher levels of sHLA-G, compared to controls. Data suggest that fetoplacental unit plays an independent role in the EPC response to a systemic autoimmune disease.

The Role of CD 34 Hematopoietic Progenitor Cells, Macrophages, and Smooth Muscle Cells in Human Coronary Artery Atherogenesis

BACKGROUND: Atherosclerosis is a widespread and devastating disease and one of the leading causes of death worldwide. So much is there to understand about atherosclerosis. And although a lot is already discovered, yet most of the studies are performed in cell cultures and animal models. Recent technologies for genetic engineering and imaging are mainly performed on animal models, with few studies in human tissues. A better understanding of their role is required.&#x0D; AIM: We aim to study the expression of CD 34 hematopoietic progenitor stem cell, CD 68 macrophages, and smooth muscle actin (SMA)-positive smooth muscle cells (SMCs) in the human coronary arteries and correlate their differential expression with the atherosclerosis progression.&#x0D; RESULTS: CD 68 and CD 34 expression increase as the atherosclerotic process proceeds from early atheroma to advanced atheroma and start to decrease as the process proceeds to fibroatheroma with a significant p &lt; 0.001. Conversely, SMA expression decreases as the atherosclerotic process progresses with a significant p &lt; 0.001.&#x0D; CONCLUSION: CD34 progenitor cells in conjunction with CD 68 macrophages have a major role in the development of atherosclerosis, whereas the SMCs are minimal in the early stages and reach their maximal levels during the stage of fibroatheroma.

Cord Blood Transplants Supported By Unrelated Donor CD34 Progenitor Cells

Abstract Introduction: Umbilical cord blood (UCB) transplant supported by third party CD34-selected cells results in rapid count recovery, UCB mediated GVL effects and low rates of chronic GvHD. For patients lacking haplo-identical relatives, other sources of CD34 progenitors are needed. Methods: We identified partially matched unrelated donors for patients lacking suitable haplo-identical donors -including second-degree relatives - and who otherwise were candidates for haplo-cord transplant. Most were treated on prospective studies (clinicaltrials.gov 00943800 and 01810588). For 5 patients, UCB were obtained through NCT01351545. Results: Between 12/2014 to 7/2018 of 126 candidates for haplo-cord transplant, 26 (21%) had no suitable related donor. Most common reasons were: no first or second degree partially matched donors, ill or unavailable relative, high titers of HLA-antibodies against all relatives, or evidence of hematological familial hereditary syndrome. Diagnoses were: 16 AML/MDS, 5 ALL, 2 MPN, 1 NHL, 1 plasma cell leukemia and 1 SAA. Median age was 57 (24-75). 50% were women. 42% non-Hispanic white (NHW), 19% non-Hispanic blacks (NHB), 19% Hispanic, 8% other minorities. CIBMTR Disease Risk Index (DRI): 46% intermediate, 38% high and 8% very high. Median HCT-CI was 3.5 (25%-75% IQR 2-6). Conditioning: 16 Flu/Mel/TBI (94% 400cGray), 8 Flu/Mel,1 Flu/Cy, 1 Etoposide/TBI. All except 1 received ATG 4.5 mg/kg and all received post-transplant Tacrolimus and MMF. UCB matching was 4/8, 5/8, 6/8 or 7/8 HLA in 12%, 27%, 23%, and 38%, respectively. HLA matching for unrelated haplos was 5-6/12, 7-8/12, &gt;9/ 12 HLA in 15%, 54% and 19%, respectively. For UCB, median cells collected were 2.1 (range1.1-4.0) ×107 TNC/kg. CD34 cell dose of unrelated donor graft was 3-5 ×106 CD34/kg. Median time to ANC engraftment was 11 days (range 9-35) and platelet engraftment was 18 days (range11-124). Chimerism on day 56 are shown in figure 1. There were three patients with graft failures: Pt #5 with complete graft rejection, autologous reconstitution, but ongoing clinical remission that persists to date (Day+ 1223); Pt #13 with graft failure and subsequent relapse, rescued with second UCB HSCT but died of progression of disease; and pt #18 who died from infectious complications after graft failure. Acute GvHD occurred in 35% of patients (4 Grade I-II, 4 Grade III, and 1 Grade IV). cGvHD was rare: One patient with mild severity and a second with severe lung involvement. After a median follow up of 209 days, 6 months cumulative incidence of relapse (CIR) was 16% and non-relapse mortality (NRM) was 20%. Median OS was 14 months (95%CI 7-27). Conclusion: 20% of adults without matched related or unrelated donors, also lack suitable first or second degree haplo-identical donors. UCB transplant with partially matched unrelated donor accessory cells provides an alternative for transplantation. Engraftment is rapid, rates of acute GVHD are acceptable and incidence of chronic GVHD is very low. Prolonged survival is encouraging in this patient cohort with adverse characteristics and who would not be candidate for haplo-identical HSCT. Disclosures No relevant conflicts of interest to declare.

Evaluation of endothelial progenitor cell (CD34) as a marker of cardiovascular risk in children on regular hemodialysis.

Endothelial progenitor cells (EPCs) CD34 are bone marrow-derived cells that decrease in chronic kidney disease (CKD) patients especially when they reach end-stage renal disease and may be a risk marker of cardiovascular (CV) diseases. The aim of our study is to investigate the endothelial progenitor cell CD 34 numbers in children with CKD on regular hemodialysis (HD) and detect their association with vascular stiffness. We recruited 25 children on regular HD, who were selected from the HD unit of Al-Zahraa Hospital, Al-Azhar University. Another group of 25 age and sex matched healthy children served as as controls. We investigated the number of EPC number (CD34) using flow cytometry, intima-media thickness (IMT), and the peak systolic velocity (PSV) of the main arteries including the (aorta, carotid, and femoral) arteries using Doppler ultrasound, this is in the same line with the routine and traditional investigations of the CV risk in the study groups. Children on regular HD have significantly lower EPC numbers (CD34 numbers) compared to their controls, the median and the inter equatorial range of CD34 was 57 (17-122) and five (3-6), respectively (P 0.001). Significant positive correlations were found between CD 34 and triglycerides serum level (r = 0.817, P = 0.001), also between CD34 with IMT and PSV of the aorta (r = 0.685, P = 0.000: r = 0.457, P = 0.022), respectively. CD34 is 88% sensitive and specific for the detection of CV risk in children on regular HD. EPC CD34 exhibited a higher predictive value for CV risk in children on regular HD. Reduced EPC numbers contribute to accelerated atherosclerosis.

Local Anesthetic Drug Inhibits Growth and Survival in Chronic Myeloid Leukemia Through Suppressing PI3K/Akt/mTOR

BackgroundApart from the known anesthetic and antiarrhythmic effects, recent studies also highlight the anticancer activities of local anesthetics. In line with the findings, our work shows that ropivacaine, an amide-linked local anesthetic drug, targets chronic myeloid leukemia (CML) via inhibiting PI3K/Akt/mTOR. Materials and MethodsThe effects of ropivacaine in CML cell lines and primary stem or progenitor cells were investigated using apoptosis, proliferation and colony formation assays. The effects of ropivacaine on proliferation and survival pathways were analyzed using Western blot. ResultsWe demonstrate that ropivacaine dose and time dependently inhibits proliferation in CML cell lines via arresting cell at G2/M stage. Ropivacaine induces apoptosis in CML cells. In addition, the anti-CML activity of ropivacaine is mainly through growth arrest rather than apoptosis induction. We further demonstrate that ropivacaine induces apoptosis and inhibits colony formation in CD34 progenitor or stem cells derived from patients with blast phase-CML. Importantly, combination of ropivacaine with imatinib or dasatinib (Bcr-Abl tyrosine kinase inhibitors) is significantly more effective in targeting CML cell lines as well as blast phase-CML CD34 cells than imatinib or dasatinib alone. We further demonstrate that ropivacaine inhibits phosphorylation of essential molecules involved in PI3K/Akt/mTOR signaling pathways in CML cells. Akt overexpression significantly reverses the effects of ropivacaine, further confirming that ropivacaine acts on CML cells via inhibition of PI3K/Akt/mTOR. ConclusionsOur work provide rationales on clinical trials for the use of local anesthetics in CML by demonstrating the anti-CML effects of ropivacaine and the molecular mechanism of its action.

Propofol enhances BCR-ABL TKIs\u2019 inhibitory effects in chronic myeloid leukemia through Akt/mTOR suppression

BackgroundThe anti-cancer activities of intravenous anesthetic drug propofol have been demonstrated in various types of cancers but not in chronic myeloid leukemia (CML).MethodsWe systematically examined the effect of propofol and its combination with BCR-ABL tyrosine kinase inhibitors (TKIs) in CML cell lines, patient progenitor cells and mouse xenograft model. We analyzed propofol’s underlying mechanism focusing on survival pathway in CML cells.ResultsWe show that propofol alone is active in inhibiting proliferation and inducing apoptosis in KBM-7, KU812 and K562 cells, and acts synergistically with imatinib or dasatinib, in in vitro cell culture system and in vivo xenograft model. In addition, propofol is more effective in inducing apoptosis and inhibiting colony formation in CML CD34 progenitor cells than normal bone marrow (NBM) counterparts. Combination of propofol and dasatinib significantly eliminates CML CD34 without affecting NBM CD34 cells. We further demonstrate that propofol suppresses phosphorylation of Akt, mTOR, S6 and 4EBP1 in K562. Overexpression of constitutively active Akt significantly reverses the inhibitory effects of propofol in K562, confirm that propofol acts on CML cells via inhibition of Akt/mTOR. Interestingly, the levels of p-Akt, p-mTOR and p-S6 are lower in cells treated with combination of propofol and imatinib than cells treated with propofol or imatinib alone, suggesting that propofol augments BCR-ABL TKI’s inhibitory effect via suppressing Akt/mTOR pathway.ConclusionOur work shows that propofol can be repurposed to for CML treatment. Our findings highlight the therapeutic value of Akt/mTOR in overcoming resistance to BCR-ABL TKI treatment in CML.

Small Molecule Based Ex Vivo Expansion of CD34+CD90+CD49f+ Hematopoietic Stem & Progenitor Cells from Non-Enriched Umbilical Cord Blood Mononucleated Cells

Umbilical cord blood (UCB) transplantation in adults have slow hematopoietic recovery compared to bone marrow (BM) or peripheral blood grafts mainly due to low number of total nucleated cells (TNC) and hematopoietic stem & progenitor cells (HSPC). Current investigational clinical strategies focus on increasing HSPC dosage by expanding CD34 enriched grafts that have resulted in early neutrophil recovery followed by long term hematopoietic reconstitution.In an effort to expand HSPC, specifically those expressing primitive phenotype (CD34+CD90+CD49f+) from non-enriched UCB, we developed a proprietary library of 50 small molecules using structure-activity-relationship studies. Freshly-thawed UCB-mononucleated cells (MNC), were cultured in serum or animal component free expansion medium supplemented with optimal concentration of respective compound. The effects of the expansion protocol were measured based on phenotypic and functional assays. Cell cultures with basal cytokines served as control.Screening of the small molecule library showed negligible acute adverse effects on CD45+ leukocyte population and its viability within 72 hours compared to cytokine control. In long term expansion cultures lasting up to 11 days, one specific structural analog, C7, resulted in 1195.8±71.7-folds increase of absolute CD45+CD34+CD38-CD45RA- progenitors which was at least 9.2-folds higher than control cultures (P<0.01; n=4). Colony forming unit assay showed significant increase of granulocyte-macrophage colonies from C7 treated cells compared to cytokine control (P<0.01; n=6) although TNC expansion was comparable between the culture conditions. It was necessary for the cytokine cocktail to comprise of at least stem cell factor, thrombopoietin and Fms-related tyrosine kinase 3 ligand for mediating HSPC expansion in presence of C7, although further addition of insulin like growth factor binding protein 2 marginally boosted expansion (P<0.001; n=3). Majority of HSPC expansion occurred between the seventh and tenth day of the culture period. In MNC initiated cultures, addition of C7 boosted primitive HSPC (CD45+CD34+CD38-CD45RA-CD90+CD49f+) by 633.3±8.5-folds over 10 days which was at least 7.4-folds higher than control cultures (P<0.001; n=3). In cultures initiated with purified CD34+CD38- cells, there was at least 15.9-folds higher expansion of HSPC defined by CD45+CD34+CD38-CD45RA-CD90+ in presence of C7 compared to cytokine cultures (P<0.05; n=3). Expansion of HSPC by C7 was at least 2-folds higher in comparison to mesenchymal stromal co-culture system which is the only known clinical protocol that allows for UCB expansion without prior CD34/CD133 selection (P<0.001; n=6). Transplantation of C7 expanded UCB grafts (n=11) at equivalent dosage of 2.5x107 cells/kg to sub-lethally irradiated NOD SCID Gamma (NSG) mice resulted in 3.21- and 2.09-folds higher engraftment of human CD45+ cells in the peripheral blood by day 21 compared to non-expanded (P=0.0030; n=6) and cytokine expanded grafts (P=0.0005; n=12) respectively. The frequency of SCID repopulating cells contributing to early peripheral blood engraftment was 2.48-folds higher in C7 expanded graft compared to unmanipulated graft. C7 expanded graft sustained human cell engraftment over 19 weeks which were primarily myeloid cells (CD33+/CD15+) as opposed to non-expanded graft which consisted of CD3+ T cells. Analysis of NSG BM at week 19 post-transplantation, showed significantly better (P<0.0001) chimerism of human CD45 cells in female (n=15) recipient compared to male (n=14) irrespective of graft type (transplantation dosage: 2.5x107 cells/kg). C7 expanded graft gave comparable level of human CD45 and CD34 progenitor cell engraftment as that of the non-expanded grafts. Multi-lineage reconstitution of NSG BM comprising of both myeloid and lymphoid human cells could be achieved with the C7 expanded graft. At higher transplantation dosage of 5.0x107 cells/kg, the expanded grafts had a higher survival rate of 75% compared to 50% for non-expanded graft mainly due to lower incidence of graft-versus-host-disease.In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment or stromal cell co-culture. The expanded UCB consists of phenotypically defined primitive HSPC that maintains in vitro and in vivo functionality. DisclosuresHwang:Celgene Corporation: Honoraria, Other: Travel Support; Roche Singapore: Honoraria, Other: Travel Support; Pfizer Singapore: Honoraria, Other: Travel Support; Novartis International AG: Honoraria, Other: Travel Support; Bristol-Myers Squibb Pte Ltd: Honoraria, Other: Travel Support; MSD Pharma (Singapore): Honoraria, Other: Travel Support; Sanofi Aventis Singapore: Honoraria, Other: Travel Support; Janssen-Cilag Singapore: Honoraria, Other: Travel Support.

Metabolism Regulates Cellular Functions of Bone Marrow-Derived Cells used for Cardiac Therapy.

Administration of bone marrow-derived mononuclear cells (BMC) may increase cardiac function after myocardial ischemia. However, the functional capacity of BMC derived from chronic heart failure (CHF) patients is significantly impaired. As modulation of the energy metabolism allows cells to match the divergent demands of the environment, we examined the regulation of energy metabolism in BMC from patients and healthy controls (HC). The glycolytic capacity of CHF-derived BMC is reduced compared to HC, whereas BMC of metabolically activated bone marrow after acute myocardial infarction reveal increased metabolism. The correlation of metabolic pathways with the functional activity of cells indicates an influence of metabolism on cell function. Reducing glycolysis without profoundly affecting ATP-production reversibly reduces invasion as well as colony forming capacity and abolishes proliferation of CD34(+) CD38(-) lin(-) hematopoietic stem and progenitor cells (HSPC). Ex vivo inhibition of glycolysis further reduced the pro-angiogenic activity of transplanted cells in a hind limb ischemia model in vivo. In contrast, inhibition of respiration, without affecting total ATP production, leads to a compensatory increase in glycolytic capacity correlating with increased colony forming capacity. Isolated CD34(+) , CXCR4(+) , and CD14(+) cells showed higher glycolytic activity compared to their negative counterparts. Metabolic activity was profoundly modulated by the composition of media used to store or culture BMC. This study provides first evidence that metabolic alterations influence the functional activity of human HSPC and BMC independent of ATP production. Changing the balance between respiration and glycolysis might be useful to improve patient-derived cells for clinical cardiac cell therapy. Stem Cells 2016;34:2236-2248.

Depletion of SAM50 Specifically Targets BCR-ABL-Expressing Leukemic Stem and Progenitor Cells by Interfering with Mitochondrial Functions.

A high proliferation rate of malignant cells requires an increased energy production, both by anaerobic glucose metabolism and mitochondrial respiration. Moreover, increased levels of mitochondria-produced reactive oxygen species (ROS) promote survival of transformed cells and contribute to the disease progression both in solid tumors and leukemia. Consequently, interfering with mitochondrial metabolism has been used as a strategy to specifically target leukemic cells. SAM50 is a mitochondrial outer membrane protein involved in the formation of mitochondrial intermembrane space bridging (MIB) complex. Although the importance of SAM50 in maintaining MIB integrity and in the assembly of mitochondrial respiratory chain complexes has been described, its specific role in the normal and leukemic hematopoietic cells remains unknown. We observed that human leukemic cells display a specific dependency on SAM50 expression, as downregulation of SAM50 in BCR-ABL-expressing, but not normal CD34(+) human hematopoietic stem and progenitor cells (HSPCs) caused a significant decrease in growth, colony formation, and replating capacity. Mitochondrial functions of BCR-ABL-expressing HSPCs were compromised, as seen by a decreased mitochondrial membrane potential and respiration. This effect of SAM50 downregulation was recapitulated in normal HSPCs exposed to cytokine-rich culture conditions that stimulate proliferation. Both oncogene-transduced and cytokine-stimulated HSPCs showed increased mitochondrial membrane potential and increased ROS levels compared to their normal counterparts. Therefore, we postulate that human leukemic HSPCs are highly dependent on the proper functioning of mitochondria and that disruption of mitochondrial integrity may aid in targeting leukemic cells.

Generation of Humanized Mice for Analysis of Human Dendritic Cells.

Transplantation of human CD34(+) hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) including dendritic cells (DCs) in vivo. Therefore, it can be a powerful tool to study human DC subsets, residing in different lymphoid and nonlymphoid organs. We have recently generated novel mouse strains called human cytokine knock-in mice in which human versions of several cytokines are knocked into Rag2(-/-)γC(-/-) strains. In addition, human SIRPα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene. These mice efficiently support human myeloid cell development and, indeed, allow the analysis of three major subsets of human DC lineages, plasmacytoid DCs and CD1c(+) and CD141(+) classical DCs. Moreover, these strains also support cytokine-mobilized peripheral blood CD34(+) cell engraftment and subsequent DC development. Here we describe our standard methods to characterize DCs developed in human cytokine knock-in mice.

Adipogenic Mesenchymal Stromal Cells from Bone Marrow and Their Hematopoietic Supportive Role: Towards Understanding the Permissive Marrow Microenvironment in Acute Myeloid Leukemia.

The role of bone marrow-derived mesenchymal stem/stromal cells (MSCs) in creating a permissive microenvironment that supports the emergence and progression of acute myeloid leukemia (AML) is not well established. We investigated the extent to which adipogenic differentiation in normal MSCs alters hematopoietic supportive capacity and we undertook an in-depth comparative study of human bone marrow MSCs derived from newly diagnosed AML patients and healthy donors, including an assessment of adipogenic differentiation capacity. MSCs from healthy controls with partial induction of adipogenic differentiation, in comparison to MSCs undergoing partial osteogenic differentiation, expressed increased levels of hematopoietic factors and induced greater proliferation, decreased quiescence and reduced in vitro hematopoietic colony forming capacity of CD34(+) hematopoietic stem and progenitor cells (HSPCs). Moreover, we observed that AML-derived MSCs had markedly increased adipogenic potential and delayed osteogenic differentiation, while maintaining normal morphology and viability. AML-derived MSCs, however, possessed reduced proliferative capacity and decreased frequency of subendothelial quiescent MSCs compared to controls. Our results support the notion of a bone marrow microenvironment characterized by increased propensity toward adipogenesis in AML, which may negatively impact normal hematopoiesis. Larger confirmatory studies are needed to understand the impact of various clinical factors. Novel leukemia treatments aimed at normalizing bone marrow niches may enhance the competitive advantage of normal hematopoietic progenitors over leukemia cells.

The Aryl Hydrocarbon Receptor Antagonist StemRegenin1 Improves In Vitro Generation of Highly Functional Natural Killer Cells from CD34(+) Hematopoietic Stem and Progenitor Cells.

Early natural killer (NK)-cell repopulation after allogeneic stem cell transplantation (allo-SCT) has been associated with reduced relapse rates without an increased risk of graft-versus-host disease, indicating that donor NK cells have specific antileukemic activity. Therefore, adoptive transfer of donor NK cells is an attractive strategy to reduce relapse rates after allo-SCT. Since NK cells of donor origin will not be rejected, multiple NK-cell infusions could be administered in this setting. However, isolation of high numbers of functional NK cells from transplant donors is challenging. Hence, we developed a cytokine-based ex vivo culture protocol to generate high numbers of functional NK cells from granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) hematopoietic stem and progenitor cells (HSPCs). In this study, we demonstrate that addition of aryl hydrocarbon receptor antagonist StemRegenin1 (SR1) to our culture protocol potently enhances expansion of CD34(+) HSPCs and induces expression of NK-cell-associated transcription factors promoting NK-cell differentiation. As a result, high numbers of NK cells with an active phenotype can be generated using this culture protocol. These SR1-generated NK cells exert efficient cytolytic activity and interferon-γ production toward acute myeloid leukemia and multiple myeloma cells. Importantly, we observed that NK-cell proliferation and function are not inhibited by cyclosporin A, an immunosuppressive drug often used after allo-SCT. These findings demonstrate that SR1 can be exploited to generate high numbers of functional NK cells from G-CSF-mobilized CD34(+) HSPCs, providing great promise for effective NK-cell-based immunotherapy after allo-SCT.

Circulating Stem Cells Associate With Adiposity and Future Metabolic Deterioration in Healthy Subjects.

Obesity and metabolic syndrome are associated with mild leukocytosis, but whether hematopoietic stem/progenitor cells (HSPCs) play a role in metabolic deterioration is unknown. Our objective was to analyze the cross-sectional and longitudinal associations between CD34(+) HSPCs, adiposity, and metabolic syndrome features. This is a cross-sectional study on 242 participants, 155 of whom were followed and included in a longitudinal assessment. This study took place in a tertiary referral center for metabolic diseases. Healthy working individuals attending a cardiovascular screening program (total n = 3158) and having a baseline measure of circulating CD34(+) cells participated. We collected demographic and anthropometric data, cardiovascular risk factors, and metabolic syndrome parameters. Participants (34.7% males, mean age 45.9 ± 0.5 years) were free from diabetes and cardiovascular disease. Cross-sectionally, absolute CD34(+) cell counts were directly correlated with body mass index and waist circumference, inversely correlated with high-density lipoprotein cholesterol and the quantitative insulin sensitivity check index, and were higher in individuals with the metabolic syndrome. The hematopoietic component contributed most to the association of CD34(+) cells with adiposity. During a 6.3-year follow-up, high absolute levels of CD34(+) cells were associated with increasing waist circumference, declining quantitative insulin sensitivity check index and high-density lipoprotein cholesterol, and with incidence of metabolic syndrome. Relative CD34(+) cell counts showed weaker associations with metabolic parameters than absolute levels, but were longitudinally associated with increasing waist circumference and metabolic syndrome development. A mild elevation of circulating CD34(+) progenitor cells, reflecting expansion of HSPCs, is associated with adiposity and future metabolic deterioration in healthy individuals.