miR-126 regulates angiogenesis in myocardial ischemia by targeting HIF-1α.

Promoting angiogenesis by targeting various angiogenic regulators has emerged as a new treatment strategy for myocardial ischemia (MI). MicroRNA-126 (miR-126) has been identified as the main regulator of compensatory angiogenesis; however, its role in MI is unclear. A rat MI model and an EA. hy926 endothelial cell hypoxia model were constructed and it was found that miR-126 was highly expressed in both models. The knockdown of HIF-1α expression in EA. hy926 cells in turn downregulated VEGF and CD34 expression and consequently inhibited angiogenesis. MiR-126 inhibitor inhibited EA. hy926 cell migration and tube formation as well as downregulated VEGF and CD34 expression, and these were reversed by transfection of miR-126 mimics. Rescue tests using miR-126 and HIF-1α demonstrated that miR-126-mediated regulation of angiogenesis was dependent on HIF-1α. In summary, miR-126 regulates the occurrence and progression of angiogenesis during MI via HIF-1α and may be a potential new therapeutic target.

Simultaneous enumeration of CD34+ and CD45+ cells using EasyCounter image cytometer.

Accurate counting of CD34-positive cells is important for successful hematopoietic stem cell transplantation that is applied to various diseases. The aim of this study was simultaneous counting of viable CD34+ (vCD34+) and CD45+ (vCD45+) cells in apheresis samples by automatic immunofluorescence counter - EasyCounter BC. CD34+ and CD45+ cells were counted using two conjugates anti-CD34 antibody - dR110 and anti-CD45 antibody - ATTO620, respectively. The conjugates were prepared by carbodiimide method. Dead nuclear cells were counted by using monomethine cyanine dye PO-TEDM 1. The linearity and reproducibility of EasyCounter BC for CD34+ cell counting were determined (R2 = 0.99; CV values for vCD34+ cells were 6.8 ÷ 8.5% and for vCD45+ cells 4.1 ÷ 7.2%). The obtained results by EasyCounter BC were compared with those by other two standard methods - flow cytometry (Guava easyCyte 8HT) and fluorescence microscopic method (Olympus BX51) with the same conjugates. Passing-Bablok regression was performed to determine the relationship between the results of the three methods, analyzing 43 apheresis samples. Correlation coefficients for vCD45+ and vCD34+ between EasyCounter BC and Olympus microscope were 0.987 and 0.982, respectively (P < 0.0001). Better results were obtained between EasyCounter BC and flow cytometer Guava, 0.998 for vCD45+ and 0.998 for vCD34+ (P < 0.0001).

Human primary CD34+ cells transplantation for critical limb ischemia.

BACKGROUND: The goal of this study was to characterize the properties of human CD34+ cells in culture and investigate the feasibility and efficacy of CD34+ transplantation in a mouse model of limb ischemia and in patients with no-option critical limb ischemia. METHODS: Human CD34+ cells isolated from peripheral blood and grown in culture for up to four passages stained positively for the surface markers CD34 and CD133 and showed high viability after cryopreservation and recovery. Seven days after surgery to induce limb ischemia, ischemic muscles of nude mice were injected with CD34+ cells. Two weeks later, mice were scored for extent of ischemic injury, and muscle tissue was collected for immunohistochemical analysis of vascular endothelial cells and RT-PCR analysis of cytokine expression. RESULTS: Injury scores of CD34+ -treated, but not control, mice were significantly different before and after transplantation. Vascular density and expression of VEGF and bFGF mRNAs were also significantly increased in the treated mice. Patients with severe lower extremity arterial ischemia were injected with their own CD34+ cells in the affected calf, foot, or toe. Significant improvements were observed in peak pain-free walking time, ankle-brachial index, and transcutaneous partial oxygen pressure. These findings demonstrate that growth of human CD34+ cells in vitro and cryopreservations are feasible. CONCLUSION: Such cells may provide a renewable source of stem cells for transplantation, which appears to be a feasible, safe, and effective treatment for patients with critical limb ischemia.

CD34-Selected Hematopoietic Stem Cell Transplants Conditioned with Myeloablative Regimens and Antithymocyte Globulin for Advanced Myelodysplastic Syndrome: Limited Graft-versus-Host Disease without Increased Relapse.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for patients with myelodysplastic syndrome (MDS). Donor T cells are critical for the graft-versus-tumor effect but carry the risk of graft-versus-host disease (GVHD). CD34 selection with immunomagnetic beads has been an effective method of depleting alloreactive donor T cells from the peripheral blood graft and has been shown to result in significant reduction in acute and chronic GVHD. We analyzed the outcomes of 102 adults (median age, 57.6 years) with advanced MDS who received a CD34-selected allo-HSCT between January 1997 and April 2012 at Memorial Sloan Kettering Cancer Center. The cumulative incidences of grades II to IV acute GVHD were 9.8% at day 100 (95% confidence interval [CI], 5.0% to 16.5%) and 15.7% at day 180 (95% CI, 9.4% to 23.4%). The cumulative incidence of chronic GVHD at 1 year was 3.9% (95% CI, 1.3% to 9.0%). The cumulative incidences of relapse were 11.8% at 1 year (95% CI, 6.4% to 18.9%) and 15.7% at 2 years (95% CI, 9.4% to 23.4%). Forty-eight patients were alive with a median follow-up of 71.7 months. Rates of overall survival (OS) were 56.9% at 2 years (95% CI, 48% to 67.3%) and 49.3% at 5 years (95% CI, 40.4% to 60.2%). Rates of relapse-free survival (RFS) were 52.0% at 2 years (95% CI, 41.9% to 61.1%) and 47.6% at 5 years (95% CI, 37.5% to 56.9%). The cumulative incidences of nonrelapse mortality were 7.8% at day 100 (95% CI, 3.7% to 14.1%), 22.5% at 1 year (95% CI, 15.0% to 31.1%), and 33.4% at 5 years (95% CI, 24.2% to 42.6%) post-transplant. The incidence of chronic GVHD/RFS overlapped with RFS. These findings demonstrate that ex vivo T cell-depleted allo-HSCT by CD34 selection offers long-term OS and RFS with low incidences of acute and chronic GVHD and without an increased risk of relapse.

Concise review: evidence for CD34 as a common marker for diverse progenitors.

CD34 is a transmembrane phosphoglycoprotein, first identified on hematopoietic stem and progenitor cells. Clinically, it is associated with the selection and enrichment of hematopoietic stem cells for bone marrow transplants. Due to these historical and clinical associations, CD34 expression is almost ubiquitously related to hematopoietic cells, and it is a common misconception that CD34-positive (CD34(+) ) cells in nonhematopoietic samples represent hematopoietic contamination. The prevailing school of thought states that multipotent mesenchymal stromal cells (MSC) do not express CD34. However, strong evidence demonstrates CD34 is expressed not only by MSC but by a multitude of other nonhematopoietic cell types including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial progenitors. In many cases, the CD34(+) cells represent a small proportion of the total cell population and also indicate a distinct subset of cells with enhanced progenitor activity. Herein, we explore common traits between cells that express CD34, including associated markers, morphology and differentiation potential. We endeavor to highlight key similarities between CD34(+) cells, with a focus on progenitor activity. A common function of CD34 has yet to be elucidated, but by analyzing and understanding links between CD34(+) cells, we hope to be able to offer an insight into the overlapping properties of cells that express CD34.

Morphometric study of nuclei and microvessels in gliomas and its correlation with grades.

INTRODUCTION: Modifications of nuclear morphology in conjunction with alteration in microvascular configuration are essential features encountered during the progression of glial tumors. In order to gain more insight into tumor biology of gliomas, objectives of the study were selected (a) to correlate morphometrically evaluated nuclear parameters [nuclear area (NA), nuclear perimeter (NP), nuclear density (ND), percentage of total nuclear area (%TNA)] and microvessel parameters [microvessel density (MVD), microvessel caliber (VC), microvessel cross sectional area (VCSA), total microvessel boundary density (TVBD), percentage of total VCSA (%TVCSA)] with WHO grading; (b) extend such correlations to the ratio parameters: ratio of MVD to ND (MDV/ND), ratio of TVBD to %TNA (TVBD/%TNA) and ratio of %TVCSA to %TNA (%TVCSA/%TNA); and (c) to correlate microvessel and ratio parameters with NP and ND. MATERIALS AND METHODS: A total of thirty gliomas managed at this institute during 2009-2012 were evaluated for various nuclear and microvessel parameters by image morphometry using a computerized digital photomicrograph system. For assessment of microvessel parameters CD34-immunostained sections were used while nuclear morphometry was performed on routine hematoxylin-eosin-stained sections. Appropriate statistical analysis was performed in correlation studies. RESULTS: All nuclear morphometric parameters showed strong positive correlation with tumor grades (r>0.7). In contrast, though all microvessel parameters exhibited positive correlation with grades, the parameters TVBD and %TVCSA showed strong positive correlation. The ratio parameters (MVD/ND) and (TVBD/%TNA) showed negative correlation with grades, whereas (%TVCSA/%TNA) did not exhibit meaningful correlation with grades. Further, while all microvessel parameters showed positive correlation with NP and ND; ratio parameters showed negative correlation with them. CONCLUSION: This study indicates that the parameters related to tumor growth (NA, NP, ND, %TNA), and angiogenesis showed increasing trend with tumor grades simultaneously; whereas the parameters related to supply of nutrients per nucleus showed decreasing trends with tumor grades, nuclear size and nuclear density. Thus, the former accounts for increased cellularity, mitosis, and vascular proliferation, while the latter culminates in tumor necrosis, all of which are essential components for grading of gliomas. The present study will therefore have a vital role as surrogate markers of grading of tumor.

Polyunsaturated fatty acids confer cryoresistance on megakaryocytes generated from cord blood and also enhance megakaryocyte production from cryopreserved cord blood cells.

BACKGROUND AIMS: Previous data have shown that the addition of docosahexanoic acid (DHA)/arachidonic acid (AA) has a beneficial effect on cytokine-mediated in vitro generation of megakaryocytes (MK) from umbilical cord blood (UCB).Cryopreservation forms an inherent part of UCB banking and MK progenitors are known to be very sensitive to the stresses of freezing. It is therefore imperative to generate functional cells from cryopreserved cells, and the generated cells need to be cryopreserved until used. In the present study, cryopreservation of ex vivo-expanded MK as well as MK generation from cryopreserved UCB samples was investigated. METHODS: MK generated with or without DHA/AA were cryopreserved in freezing medium containing 10% dimethyl sulfoxide (DMSO). Freezing efficacy was tested by quantitating MK after revival. Cryopreserved CD34(+) cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO), in the presence and absence of DHA/AA for 10 days, and then quantitated for MK. Results. We observed a 1.5-3-fold increase in MK numbers, their progenitor content and their expression of phenotypic markers and MK-related transcription factors. DHA/AA sets showed a 2-5-fold improved engraftment in NOD/SCID mice. These data showed that the beneficial effect of DHA/AA obtained during MK expansion was not altered after freezing stress. The enhancement in MK generation obtained from fresh cord blood (CB) cells was reproduced with comparable efficiency when we used cryopreserved CB samples. CONCLUSIONS: Taken together, our data suggest that in vitro-generated DHA/AA MK survive cryoinjuries in a functionally better state. DHA/AA support a more efficient generation of MK from cryopreserved UCB.

Glycoforms of human endothelial CD34 that bind L-selectin carry sulfated sialyl Lewis x capped O- and N-glycans.

Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLe(x)). It is thought that multivalent 6-sulfo SLe(x) expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLe(x) in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin-binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLe(x) epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLe(x). N-glycans containing potential 6-sulfo SLe(x) epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLe(x) epitopes on O-glycans that are important for its recognition by L-selectin.

Transportation of cellular therapy products: report of a survey by the cellular therapies team of the Biomedical Excellence for Safer Transfusion (BEST) collaborative.

BACKGROUND AND OBJECTIVES: Most cell therapy products (CTP) are infused or processed shortly after collection but in some cases this may be delayed for up to 48 h. A number of variables such as temperature and cell concentration are of critical importance for the integrity of CTP during this time. MATERIALS AND METHODS: We conducted a survey of cellular therapy laboratories to ascertain current practices for CTP transportation. RESULTS: There were 194 respondents of whom 90% shipped or received CTP--84% allogeneic, 71% autologous and 62% therapeutic cells. Processing facilities shipped or received the following products--hematopoietic progenitor cells (HPC), Marrow 73%; HPC, Apheresis 90%; HPC, Cord Blood 54% and others 14%. Other CTP included donor lymphocytes, mesenchymal stem cells (MSC), natural killer cells, buffy coat neutrophils and virus-specific cytotoxic T lymphocytes (CTL). More than 70% of respondents believed that it was acceptable for CTP to be held for up to 2 h without checking the temperature or cell density and a similar proportion agreed that putting products in containers to control parameters such as temperature within this time period was unnecessary. The majority of centres shipped or received between 1 and 10 CTP annually and 66% received products taking more than 2 h to ship. Of these, 82% specified the conditions for temperature in transit whilst 57% monitored temperature in transit and 74% of these used a data logger. The temperature range most commonly specified was 18-24 degrees C. The majority of processing facilities did not request an adjustment to the cell density even for products taking more than 2 h to reach their facility. More than 90% of respondents tested HPC for CD34(+) cells, viability and sterility; 40-48% performed colony-forming unit-granulocyte macrophage (CFU-GM) analysis. Only viability was thought by > 50% of respondents to be impacted by temperature, cell density and other parameters. CONCLUSION: Understanding current practice will help in the design of future studies for CTP storage and transportation.

Sequential CD34 cell fractionation by magnetophoresis in a magnetic dipole flow sorter.

Cell separation and fractionation based on fluorescent and magnetic labeling procedures are common tools in contemporary research. These techniques rely on binding of fluorophores or magnetic particles conjugated to antibodies to target cells. Cell surface marker expression levels within cell populations vary with progression through the cell cycle. In an earlier work we showed the reproducible magnetic fractionation (single pass) of the Jurkat cell line based on the population distribution of CD45 surface marker expression. Here we present a study on magnetic fractionation of a stem and progenitor cell (SPC) population using the established acute myelogenous leukemia cell line KG-1a as a cell model. The cells express a CD34 cell surface marker associated with the hematopoietic progenitor cell activity and the progenitor cell lineage commitment. The CD34 expression level is approximately an order of magnitude lower than that of the CD45 marker, which required further improvements of the magnetic fractionation apparatus. The cells were immunomagnetically labeled using a sandwich of anti-CD34 antibody-phycoerythrin (PE) conjugate and anti-PE magnetic nanobead and fractionated into eight components using a continuous flow dipole magnetophoresis apparatus. The CD34 marker expression distribution between sorted fractions was measured by quantitative PE flow cytometry (using QuantiBRITE PE calibration beads), and it was shown to be correlated with the cell magnetophoretic mobility distribution. A flow outlet addressing scheme based on the concept of the transport lamina thickness was used to control cell distribution between the eight outlet ports. The fractional cell distributions showed good agreement with numerical simulations of the fractionation based on the cell magnetophoretic mobility distribution in the unsorted sample.

Mesenchymal stem cell-derived microvesicles mediate BMP2 gene delivery and enhance bone regeneration.

A demineralized bone matrix (DBM) scaffold has good biocompatibility, low antigenicity, a natural porous structure and no cytotoxicity, and so it is an appropriate material for bone regeneration. However, osteoinductive growth factors are often removed during preparation, which destroys the osteoinductive capacity of the DBM scaffold. Biomaterials combined with gene therapy is a promising approach to effectively avoid this adverse side effect. This study develops a human bone morphogenetic protein 2 (hBMP2) gene-activated DBM scaffold to enhance the osteoinductive capacity of DBM and improve bone repair. Bone marrow mesenchymal stem cell (MSC)-derived microvesicles (MVs) were obtained, and polyethyleneimine (PEI) and human bone morphogenetic protein 2 (hBMP2) plasmids (phBMP2) were sequentially coated on the MVs by layer-by-layer (LBL) self-assembly to form an MVs-PEI/phBMP2 non-viral gene vector. Finally, the gene-activated scaffold (DBM/MVs-PEI/phBMP2) was prepared by loading MVs-PEI/phBMP2 onto a DBM scaffold. The experimental results show that the MVs-PEI/phBMP2 exhibits higher transfection efficiency and lower cytotoxicity to MSCs when the MVs/PEI weight ratio = 5, and could enhance the osteogenic differentiation of MSCs in vitro. Subcutaneous implantation into rats showed that the DBM/MVs-PEI/phBMP2 scaffold could efficiently enhance the deposition of: collagen fibers, osteocalcin, osteopontin and CD34 endogenous proteins. Rabbit femoral condyle defect experiments proved that the DBM/MVs-PEI/phBMP2 scaffold could significantly promote bone repair. This study presents a novel, highly efficient and low cytotoxicity gene delivery vector based on MVs. The gene-activated DBM scaffold based on MVs not only could promote bone formation but also angiogenesis, implying that this kind of gene-activated scaffold is a promising bone substitute material.

ADS-J1 inhibits human immunodeficiency virus type 1 entry by interacting with the gp41 pocket region and blocking fusion-active gp41 core formation.

We previously identified a small-molecule anti-human immunodeficiency virus type 1 (anti-HIV-1) compound, ADS-J1, using a computer-aided molecular docking technique for primary screening and a sandwich enzyme-linked immunosorbent assay (ELISA) as a secondary screening method. In the present study, we demonstrated that ADS-J1 is an HIV-1 entry inhibitor, as determined by a time-of-addition assay and an HIV-1-mediated cell fusion assay. Further mechanism studies confirmed that ADS-J1 does not block gp120-CD4 binding and exhibits a marginal interaction with the HIV-1 coreceptor CXCR4. However, ADS-J1 inhibited the fusion-active gp41 core formation mimicked by peptides derived from the viral gp41 N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), as determined by ELISA, native polyacrylamide gel electrophoresis, and circular dichroism analysis. Moreover, using a surface plasmon resonance assay, we found that ADS-J1 could bind directly to IQN17, a trimeric peptide containing the gp41 pocket region, resulting in the conformational change of IQN17 and the blockage of its interaction with a short D peptide, PIE7. The positively charged residue (K574) located in the gp41 pocket region is critical for the binding of ADS-J1 to NHR. These results suggest that ADS-J1 may bind to the viral gp41 NHR region through its hydrophobic and ionic interactions with the hydrophobic and positively charged resides located in the pocket region, subsequently blocking the association between the gp41 NHR and CHR regions to form the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry.

CD34 is a key regulator of hematopoietic stem cell trafficking to bone marrow and mast cell progenitor trafficking in the periphery.

CD34 is a cell-surface sialomucin widely used for hematopoietic stem cell purification and as a marker of most vascular endothelial cells, including those of capillaries in the majority of tissues. Surprisingly, despite extensive research, the function of this sialomucin has remained elusive, with proposed roles ranging from enhancing proliferation or inhibiting differentiation to acting as a proadhesive L-selectin ligand. Here, we review our recent studies, which suggest that CD34 does, indeed, play a role in leukocyte and HSC trafficking, but that this is through its action as a regulated blocker of cell adhesion and enhancer of migration.

The C-terminal SH3 domain of CRKL as a dynamic dimerization module transiently exposing a nuclear export signal.

CRKL plays essential roles in cell signaling. It consists of an N-terminal SH2 domain followed by two SH3 domains. SH2 and SH3N bind to signaling proteins, but the function of the SH3C domain has remained largely enigmatic. We show here that the SH3C of CRKL forms homodimers in protein crystals and in solution. Evidence for dimer formation of full-length CRKL is also presented. In the SH3C dimer, a nuclear export signal (NES) is mostly buried under the domain surface. The same is true for a monomeric SH3C obtained under different crystallization conditions. Interestingly, partial SH3 unfolding, such as occurs upon dimer/monomer transition, produces a fully-accessible NES through translocation of a single beta strand. Our results document the existence of an SH3 domain dimer formed through exchange of the first SH3 domain beta strand and suggest that partial unfolding of the SH3C is important for the relay of information in vivo.

The molecular phenotypes of ureteral telocytes are layer-specific.

Telocytes (TC) are the delicate interstitial (stromal) cells defined by their long, thin and moniliform processes termed telopodes. Numerous studies determined that different subsets of telocytes populate almost all tissues and attempted to relate these subsets to various functions, from cell signaling to tissue repair and regeneration. Extremely few studies addressed the urinary tract though few data on the molecular pattern of the urinary TCs actually exist. We therefore hypothesized that subsets of urinary TCs co-localize within the human ureter and we aimed at performing an immunohistochemical study to evaluate the tissue-specific molecular pattern of TCs. On sample tissues of proximal ureter drawn from ten human adult patients during surgery were applied primary antibodies against CD34, CD105, von Willebrand Factor, the heavy chain of smooth muscle myosin (SMM) and c-erbB-2. The molecular pattern indicated three different subsets of ureteral TCs which are neither endothelial nor epithelial in nature: (a) type I: the CD34-/CD105+ TCs of the superficial layer of lamina propria; (b) type II: the CD34+/CD105± myoid TCs of the deep layer of lamina propria and (c) type III: the CD34+/CD105+ perivascular TCs. Although apparently different, all these subsets of TCs could belong to the stem/progenitor niche of the ureter.

Telocytes in human fetal skeletal muscle interstitium during early myogenesis.

A new peculiar stromal cell type called telocyte (TC)/CD34-positive stromal cell (i.e. cell with distinctive prolongations named telopodes) has recently been described in various tissues and organs, including the adult skeletal muscle interstitium of mammals. By forming a resident stromal three-dimensional network, TCs have been suggested to participate in different physiological processes within the skeletal muscle tissue, including homeostasis maintenance, intercellular signaling, tissue regeneration/repair and angiogenesis. Since a continuous interplay between the stromal compartment and skeletal muscle fibers seems to take place from organogenesis to aging, the present study was undertaken to investigate for the first time the presence of TCs in the human skeletal muscle during early myogenesis. In particular, we describe the morphological distribution of TCs in human fetal lower limb skeletal muscle during early stages of myogenesis (9-12 weeks of gestation). TCs were studied on tissue sections subjected to immunoperoxidase-based immunohistochemistry for CD34. Double immunofluorescence was further performed to unequivocally differentiate TCs (CD34-positive/CD31-negative) from vascular endothelial cells (CD34-positive/CD31-positive). Our findings provide evidence that stromal cells with typical morphological features and immunophenotype of TCs are present in the human skeletal muscle during early myogenesis, revealing differences in either CD34 immunopositivity or TC numbers among different gestation ages. Specifically, few TCs weakly positive for CD34 were found between 9 and 9.5 weeks. From 10 to 11.5 weeks, TCs were more numerous and strongly reactive and their telopodes formed a reticular network in close relationship with blood vessels and primary and secondary myotubes undergoing separation. On the contrary, a strong reduction in the number and immunopositivity of TCs was observed in fetal muscle sections from 12 weeks of gestation, where mature myotubes were evident. The muscle stroma showed parallel changes in amount, density and organization from 9 to 12 weeks. Moreover, blood vessels appeared particularly numerous between 10 and 11.5 weeks. Taken together, our findings suggest that TCs might play a fundamental role in the early myogenetic period, possibly guiding tissue organization and compartmentalization, as well as angiogenesis and maturation of myotubes.

Chimeric protein for selective cell attachment onto cellulosic substrates.

We have developed a fusion protein (CBD-LG) incorporating a cellulose-binding domain and an antibody binding domain, protein LG, to provide an adaptor molecule for cell separation with regenerated cellulose hollow fiber arrays. A single hollow fiber cell adhesion assay utilizing a CD34+ cell line, KG1a, was used to investigate whether ligand affinity interactions were strong enough for cell attachment and separation. CBD-LG efficiently captured CD34+ cells labeled with the mouse IgG2a monoclonal antibody MHCD3400. However, it was not possible to bind CD34+ cells labeled with an IgG1 antibody (HPCA-2). The low affinity of HPCA-2 for LG was overcome by secondary antibodies: KG1a cells that were dual labeled with HPCA-2 followed by rat anti-mouse IgG1 adhered inside hollow fibers coated with CBD-LG. Alternatively, immobilized rabbit polyclonal anti-mouse IgG1 captured cells labeled with HPCA-2. The development of an adaptor molecule to display recombinant domains at the surface of hollow fibers will be an effective tool to investigate cellular ligand-receptor interactions, a necessary step in the development of hollow fiber bioreactors for manufacture of human cellular products.

Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease.

Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus(+) HSPCs with 6-16% human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 resulted in ∼15% gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs, 4-11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.

Three-dimensional structure prediction of the interaction of CD34 with the SH3 domain of Crk-L.

The monomeric 115-kDa surface protein CD34, which is present on many stem cell populations, has been useful to enumerate the quality and viability of cell suspensions for engraftment. Although these studies assure the validity of CD34 as a stem cell marker, the functional role of this molecule has not been defined. CD34 has been demonstrated to regulate adhesion, differentiation, and proliferation of hematopoietic stem cells and other progenitors. The cytoplasmic domain of CD34 is known to be essential for its function. However, it is not clear how this domain's interactions with other molecules support the functional activity of CD34. Here we show that the cytoplasmic tail of CD34 is structurally similar to the carboxyl terminus of the gap junction protein Connexin 43 (Cx43). Because the activity of CD34 is mediated through its interaction with an SH3 domain of an intracellular protein, we attempted to define the SH3 binding region and amino acids involved in this interaction. We identified Glu325 to Ser334 as potential SH3 binding sites. Our results suggest that the interaction of the cytoplasmic tail of CD34 with the shallow proline-rich motif-binding groove of Crk-L is essential for the function of CD34 in stem cell development.