Neutrophils cultured ex vivo from CD34+ stem cells are immature and genetically tractable.

BACKGROUND: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development. METHODS: We optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors. RESULTS: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis. CONCLUSION: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.

Unraveling Hematotoxicity of α-Amanitin in Cultured Hematopoietic Cells.

Amanita phalloides poisonings account for the majority of fatal mushroom poisonings. Recently, we identified hematotoxicity as a relevant aspect of Amanita poisonings. In this study, we investigated the effects of the main toxins of Amanita phalloides, α- and ß-amanitin, on hematopoietic cell viability in vitro. Hematopoietic cell lines were exposed to α-amanitin or ß-amanitin for up to 72 h with or without the pan-caspase inhibitor Z-VAD(OH)-FMK, antidotes N-acetylcysteine, silibinin, and benzylpenicillin, and organic anion-transporting polypeptide 1B3 (OATP1B3) inhibitors rifampicin and cyclosporin. Cell viability was established by trypan blue exclusion, annexin V staining, and a MTS assay. Caspase-3/7 activity was determined with Caspase-Glo assay, and cleaved caspase-3 was quantified by Western analysis. Cell number and colony-forming units were quantified after exposure to α-amanitin in primary CD34+ hematopoietic stem cells. In all cell lines, α-amanitin concentration-dependently decreased viability and mitochondrial activity. ß-Amanitin was less toxic, but still significantly reduced viability. α-Amanitin increased caspase-3/7 activity by 2.8-fold and cleaved caspase-3 by 2.3-fold. Z-VAD(OH)-FMK significantly reduced α-amanitin-induced toxicity. In CD34+ stem cells, α-amanitin decreased the number of colonies and cells. The antidotes and OATP1B3 inhibitors did not reverse α-amanitin-induced toxicity. In conclusion, α-amanitin induces apoptosis in hematopoietic cells via a caspase-dependent mechanism.

Discrepancy in transcriptomic profiling between CD34 + stem cells and primary bone marrow cells in myelodysplastic neoplasm.

Differentially expressed genes (DEGs) biomarkers can be used to help diagnose and monitor the disease, as well as to determine which treatments are most effective. So, given the complexity of Myelodysplastic neoplasm (MDS), it is difficult to determine the impact and disparities of DEGs between CD34+ HSC (hematopoietic stem cells) or primary bone marrow cells (PBMC) in MDS pathogenesis, and therefore it remains largely unknown. Here, we performed an in-silico transcriptome analysis on CD34+ HSC and PBMC from 1092 MDS patients analyzing the divergences between differential gene expression patterns in these two cell types as potential pathogenic biomarkers for MDS. Initially, we observed a difference of 7117 expressed transcripts between PBMC (n = 40,165) and CD34 +HSC (n = 33,048). Also, we identified that CD34+ HSC and PBMC samples showed 240 and 2948 DEGs, respectively. In summary, we identified DEGs disparities in CD34+ HSC and PBMC cell types. However, there was a certain similarity of the activated pathways in both cellular samples based on Gene Ontology and KEGG pathways enrichment analyses. Our results provide novel insights into novel DEGs biomarkers to MDS pathogenesis with clinical significance. AVAILABILITY OF DATA AND MATERIALS: All microarray databases were obtained from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). To evaluate the biological function of differentially expressed genes, the DAVID (Database for Annotation, Visualization and Integrated Discovery tool was used) (https://david.ncifcrf.gov/).

Histological study of the role of CD34+ stem cells and mast cells in cyclophosphamide-induced thymic injury in rats and the possible attenuating role of melatonin.

Cyclophosphamide (CP) is an anticancer drug that adversely affects immunity and thymus structure. Melatonin is a hormone secreted by the pineal gland. It boosts immunity and has antioxidant properties. Therefore, the present study was conducted to investigate the possible protective effect of melatonin on CP-induced changes in the rat thymus. Forty male albino rats were used and divided equally into four main groups. Group I was the control group. Group II (melatonin group) received melatonin at a dose of 10 mg/kg body weight/day by intraperitoneal injection throughout the experimental period. Group III (CP group) received 200 mg/kg body weight CP by a single intraperitoneal injection. Group IV (CP + melatonin group) received melatonin intraperitoneally at a dose of 10 mg/kg body weight/day starting 5 days prior to CP injection until the end of the experiment. All rats were euthanized 7 days after CP injection. Administration of CP in group III resulted in depletion of the cortical thymoblasts. In addition, CD34-immunopositive stained stem cells decreased and mast cell infiltration increased. Electron microscopy showed degeneration of thymoblasts and vacuolization of epithelial reticular cells. Administration of melatonin with CP in group IV showed considerable protection of thymic histology. In conclusion, melatonin may protect against CP-induced thymic injury.

Growth hormone enhances the CD34+ stem cells repopulation of the male albino rat thymus gland in cyclophosphamide induced injury: immunohistochemical and electron microscopic study.

Cyclophosphamide (CP) is a chemotherapeutic drug that has a harmful effect on the immune system. Growth hormone (GH) is a peptide hormone that can enhance thymic functions in cases of immunosuppression. Therefore, the present study was performed to study the possible protective effect of growth hormone on cyclophosphamide-induced changes in the rat thymus gland. Sixty-four adult male albino rats were used and divided into three main groups. Group I (Control group). Group II (CP group) received 200 mg/kg body weight CP by a single intra-peritoneal injection. Group III (CP& GH group) received GH in a dose of 2 mg/kg body weight/day by subcutaneous injection starting 5 days before cyclophosphamide injection till the end of the experiment. Administration of CP (Group II) resulted in marked histopathological changes in thymus. Thymic cortex showed depletion of thymoblasts. There was a decrease in CD34 immune positively stained stem cells and an increase in CD68 immune positively stained macrophages. Ultrastructurally, thymoblasts were markedly degenerated and the most of epithelial reticular cells were vacuolated. Administration of GH (group III) showed preservation of the histological structure of the thymus. In conclusion, growth hormone could protect against cyclophosphamide induced thymic damage.

Can mobilization of bone marrow stem cells be an alternative regenerative therapy to stem cell injection in a rat model of chronic kidney disease?

Chronic kidney disease (CKD) is a priority health problem affecting 36% of Egyptians. Adipose-derived mesenchymal stem cells (ADMSCs) have multidifferentiation capacity and the ability to restore several types of cells including damaged renal cells. Granulocyte colony-stimulating factor (G-CSF) is known to mobilize hematopoietic stem cells from bone marrow to the peripheral circulation. The aim of this study was to compare the effect of endogenous CD34+ cells mobilization and exogenous ADMSCs administration in the treatment of a rat model of adriamycin (ADR)-induced CKD. A total of 48 male albino rats of the local strain (200 ± 50 g) were equally divided into four groups: control negative, ADR (control positive), ADMSCs group, and G-CSF group. Six rats from each group were sacrificed after 4 weeks and the other 6 after 12 weeks. Renal function was assessed frequently by measuring serum creatinine, albumin, urea, 24-h urinary protein level, and hemoglobin level throughout the study. Oxidative stress markers malondialdehyde (MDA) and total antioxidant (TAO) were measured on day 28. CD-34+ cell percentage was measured on day 9. After the sacrification of the rats, kidneys were removed for histopathological assessment. Results revealed that both ADMSCs and G-CSF significantly improved serum creatinine, albumin, urea, 24-h urinary protein level, and histopathological damage score, with the G-CSF-treated group showing better improvement in 24-h urinary protein level, serum albumin, and histopathological damage score compared with ADMSCs-treated group. The G-CSF group also had significantly higher levels of CD34+ cells. Oxidative stress markers (MDA and TAO) levels were significantly improved with both therapies. We conclude that mobilization of endogenous hematopoietic stem cells by G-CSF is more effective than exogenously injected ADMSCs in protecting the kidneys against AD-induced toxicity.

CRISPR-Edited Stem Cell Transplantation for HIV-Related Gene Modification In Vivo: A Systematic Review.

BACKGROUND: CRISPR is a novel genomic editing technology which can be useful for the treatment of immune diseases such as HIV. However, the application of CRISPR in stem cells for HIV-related research was not effective, and most of the research was done in vivo. This systematic review is to identify a new research idea about increase CRISPR-editing efficiencies in stem cell transplantation for HIV treatment, as well as its future perspective. METHOD: Four databases were searched for articles published during 1952 to 2020. PRISMA method was used to select appropriate research papers. CAMARADES was used to identify the paper quality. The outcome was engraftment efficiency, gene disruption percentage, differentiation ability, HIV-resistant efficiency. RESULT: Screening method showed 196 papers mentioned the topic. However, only 5 studies were reliable with the research objective. We found that (1) Two research ideas which was double gene knockout and knockout-knockin method to provide HIV-resistant cells, engraftment support and avoid cardiac disease as an HIV disease side effect. (2) Ribonucleoprotein (RNP) delivery was the best way to deliver the CRISPR/Cas9 and Adeno-Associated Virus (AAV) would be effective for knockin purpose. (3) CRISPR/SaCas9 could replace CRISPR/Cas9 role in editing HIV-related gene. CONCLUSION: Potential genes to increase HIV resistance and stem cell engraftment should be explored more in the future. Double knockout and knock-in procedures should be applied to set up a better engraftment for improving HIV treatment or resistance of patients. CRISPR/SaCas9 and RNP delivery should be explored more in the future. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020203312.

Analysis of the retinal capillary plexus layers in a murine model with diabetic retinopathy: effect of intravitreal injection of human CD34+ bone marrow stem cells.

BACKGROUND: Diabetic retinopathy is a retinal vasculopathy involving all three retinal capillary plexus layers. Since human CD34+ bone marrow stem cells (BMSCs) have the potential to promote revascularization of ischemic tissue, this study tests the hypothesis that intravitreal injection of human CD34+ BMSCs can have protective effects on all layers of the retinal vasculature in eyes with diabetic retinopathy. METHODS: Streptozotocin (STZ)-induced diabetic mice were injected intravitreally with 50,000 human CD34+ BMSCs or phosphate-buffered saline (PBS) into the right eye. Systemic immunosuppression with rapamycin and tacrolimus was started 5 days before the injection and maintained for study duration to prevent rejection of human cells. All mice were euthanized 4 weeks after intravitreal injection; both eyes were enucleated for retinal flat mount immunohistochemistry. The retinal vasculature was stained with Isolectin-GS-IB4. Confocal microscopy was used to image four circular areas of interest of retina, 1-mm diameter around the optic disc. Images of superficial, intermediate, and deep retinal capillary plexus layers within the areas of interest were obtained and analyzed using ImageJ software with the Vessel Analysis plugin to quantitate the retinal vascular density and vascular length density in the three plexus layers. RESULTS: Three distinct retinal capillary plexus layers were visualized and imaged using confocal microscopy. Eyes that received intravitreal injection of CD34+ BMSCs (N=9) had significantly higher vascular density and vascular length density in the superficial retinal capillary plexus when compared to the untreated contralateral eyes (N=9) or PBS treated control eyes (N=12; P values <0.05 using ANOVA followed by post-hoc tests). For the intermediate and deep plexus layers, the difference was not statistically significant. CONCLUSIONS: The protective effect of intravitreal injection of the human CD34+ BMSCs on the superficial retinal capillary plexus layers is demonstrated using confocal microscopy in this murine model of diabetic retinopathy.

Plerixafor combined with G-CSF for stem cell mobilization in children qualified for autologous transplantation- single center experience.

Failure of autologous peripheral blood CD34+ stem cells collection can adversely affect the treatment modality for patients with hematological and nonhematological malignant diseases where high dose chemotherapy followed by hematopoietic stem cell transplantation has become part of their treatment. Plerixafor in conjunction with G-CSF is approved for clinical use as a mobilization agent. The clinical efficacy of Plerixafor in CD34+ cells collection was analyzed in our institution. A total of 13 patients aged 1-15,5 years received Plerixafor in combination with G-CSF: 7 with neuroblastoma, 2 with Ewing's sarcoma and single patients with Hodgkin's lymphoma, germ cell tumor, retinoblastoma and Wilms tumor. Twelve patients (923%) achieved CD34+ cell counts of ≥ 20 × 106/L after 1-7 doses of Plerixafor. The average 9,9 - fold increase in number of CD34+ cells were achieved following the first dose and 429 - fold after second dose of plerixafor. Among the 13 patients, 12 yielded the minimum required cell collection of 2 × 106/kg within an average of 2 doses of Plerixafor. The mean number of apheresis days was 1.75. The median total number of collected CD34+ cells was 982 × 106/kg. Plerixafor enables rapid and effective mobilization, and collection of sufficient number of CD34+ cells.

Generation of Acute Hind Limb Ischemia in NOD/SCID Mice and Treatment with Nanofiber-Expanded CD34+ Hematopoietic Stem Cells.

Critical limb ischemia (CLI) is primarily associated with a high risk of major amputation, cardiovascular events, and death. The current therapy involves direct endovascular intervention and is associated with long-term recurrence. However, patients with significant comorbidities are not eligible for this therapy. Hind limb ischemia model via femoral artery excision has commonly been used to determine therapeutic potential and for investigating cellular and molecular mechanisms. This protocol describes the ischemic model development in NOD/SCID mice and the use of human umbilical cord blood-derived and nanofiber scaffold-expanded CD34+ stem cells to investigate the efficacy of regenerative therapy.

Generation of Myocardial Ischemic Wounds and Healing with Stem Cells.

Myocardial ischemia is a common manifestation of cardiovascular diseases (CVD) that affects the health and lives of millions of people worldwide. While numerous treatment options exist that address cardiac damage after ischemic injury, none of these can repair damaged cardiac tissue. Stem cell-mediated therapy is an emerging approach for cardiac tissue regeneration that has shown promise in preclinical models and in clinical studies. However, much more research in this field must be carried out to bring effective stem cell therapies to clinical settings. This protocol discusses the methods for generation of an animal model of myocardial ischemia in a preclinical setting, expansion of viable hematopoietic stem cells on a nanofiber scaffold, and administration of cells into the ischemic animal to verify therapeutic efficacy.

Human CD34+ Hematopoietic Stem Cell-Engrafted NSG Mice: Morphological and Immunophenotypic Features.

Immunodeficient mice engrafted with human immune cells represent an innovative tool to improve translatability of animal models for the study of human diseases. Immunophenotyping in these mice focuses on engraftment rates and cellular differentiation in blood and secondary lymphoid organs, and is predominantly carried out by FACS (fluorescent activated cell sorting) analysis; information on the morphological aspects of engraftment and the prevalence of histologic lesions is limited. We histologically examined 3- to 6-month-old NSG mice, naïve or engrafted with CD34+ human hemopoietic stem cells (HSC), and employed a quantitative immunohistochemical approach to identify human and murine cell compartments, comparing the results with the FACS data. NSG mice mainly exhibited incidental findings in lungs, kidneys, testes, and adrenal glands. A 6-month-old NSG mouse had a mediastinal lymphoblastic lymphoma. The lymphoid organs of NSG mice lacked typical lymphoid tissue architecture but frequently exhibited small periarteriolar leukocyte clusters in the spleen. Mice engrafted with human HSC frequently showed nephropathy, ovarian atrophy, cataract, and abnormal retinal development, lesions considered secondary to irradiation. In addition, 20% exhibited multisystemic granulomatous inflammatory infiltrates, dominated by human macrophages and T cells, leading to the observed 7% mortality and morbidity. Immunophenotypic data revealed variable repopulation of lymphoid organs with hCD45+ human cells, which did not always parallel the engraftment levels measured via FACS. The study describes the most common pathological features in young NSG mice after human HSC engraftment. As some of these lesions contribute to morbidity, morphological assessment of the engraftment at tissue level might help improve immunophenotypic evaluations of this animal model.

CD34+ cell therapy significantly reduces adverse cardiac events, health care expenditures, and mortality in patients with refractory angina.

Patients with refractory angina who are suboptimal candidates for further revascularization have improved exercise time, decreased angina frequency, and reduced major adverse cardiac events with intramyocardial delivery of CD34+ cells. However, the effect of CD34+ cell therapy on health care expenditures before and after treatment is unknown. We determined the effect of CD34+ cell therapy on cardiac-related hospital visits and costs during the 12 months following stem cell injection compared with the 12 months prior to injection. Cardiac-related hospital admissions and procedures were retrospectively tabulated for patients enrolled at one site in one of three double-blinded, placebo-controlled CD34+ trials in the 12 months before and after intramyocardial injections of CD34+ cells vs placebo. Fifty-six patients were randomized to CD34+ cell therapy (n = 37) vs placebo (n = 19). Patients randomized to cell therapy experienced 1.57 ± 1.39 cardiac-related hospital visits 12 months before injection, compared with 0.78 ± 1.90 hospital visits 12 months after injection, which was associated with a 62% cost reduction translating to an average savings of $5500 per cell therapy patient. Patients in the placebo group also demonstrated a reduction in cardiac-related hospital events and costs, although to a lesser degree than the CD34+ group. Through 1 January 2019, 24% of CD34+ subjects died at an average of 6.5 ± 2.4 years after enrollment, whereas 47% of placebo patients died at an average of 3.7 ± 1.9 years after enrollment. In conclusion, CD34+ cell therapy for subjects with refractory angina is associated with improved mortality and a reduction in hospital visits and expenditures for cardiac procedures in the year following treatment.

CD34 cells in somatic, regenerative and cancer stem cells: Developmental biology, cell therapy, and omics big data perspective.

The transmembrane phosphoglycoprotein protein CD34 has conventionally been regarded as a marker for hematopoietic progenitors. Its expression on these cells has been leveraged for cell therapy applications in various hematological disorders. More recently, the expression of CD34 has also been reported on cells of nonhematopoietic origin. The list includes somatic cells such as endothelial cells, fibrocytes and interstitial cells and regenerative stem cells such as corneal keratocytes, muscle satellite cells, and muscle-derived stem cells. Furthermore, its expression on some cancer stem cells (CSCs) has also been reported. Till date, the functional roles of this molecule have been implicated in a multitude of cellular processes including cell adhesion, signal transduction, and maintenance of progenitor phenotype. However, the complete understanding about this molecule including its developmental origins, its embryonic connection, and associated functions is far from complete. Here, we review our present understanding of the structure and putative functions of the CD34 molecule based upon our literature survey. We also probed various biological databases to retrieve data related to the expression and associated molecular functions of CD34. Such information, upon synthesis, is hence likely to provide the suitability of such cells for cell therapy. Moreover, we have also covered the existing cell therapy and speculated cell therapy applications of CD34+ cells isolated from various lineages. We have also attempted here to speculate the role(s) of CD34 on CSCs. Finally, we discuss number of large-scale proteomics and transcriptomics studies that have been performed using CD34+ cells.

Exosomes derived from human CD34+ stem cells transfected with miR-26a prevent glucocorticoid-induced osteonecrosis of the femoral head by promoting angiogenesis and osteogenesis.

BACKGROUND: Damaged endothelial cells and downregulated osteogenic ability are two key pathogenic mechanisms of glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH). Recent studies suggested that transplantation of CD34+ stem cell-derived exosomes (CD34+-Exos) can treat ischemic diseases by promoting neovascularization and that miR-26a is an important positive regulator of osteogenesis. Moreover, the biological effect of exosomes is closely related to their cargo miRNAs. However, it is not clear whether increasing the abundance of miR-26a in CD34+-Exos will inhibit the progress of GC-induced ONFH. METHODS: MiR-26a was overexpressed in CD34+-Exos (miR-26a-CD34+-Exos) to increase their osteogenic potential. The angiogenic potential of miR-26a-CD34+-Exos was then examined through evaluations of migration and tube-forming capacities in vitro. In addition, in order to observe the osteogenic effect of miR-26a-CD34+-Exos on bone marrow stromal cells (BMSCs), Alizarin red staining, alkaline phosphatase (ALP) activity assays, and qPCR were carried out. Finally, miR-26a-CD34+-Exos were injected into a GC-induced ONFH rat model to prevent the progress of GC-induced ONFH. The biological effects of miR-26a-CD34+-Exos on the ONFH model were evaluated by micro-CT, angiography, and histological staining. RESULTS: Our data showed that miR-26a-CD34+-Exos enhanced human umbilical vein endothelial cell migration and tube-forming capacities. Furthermore, miR-26a-CD34+-Exos strengthened the osteogenic differentiation of BMSCs under the influence of GCs in vitro. Finally, the miR-26a-CD34+-Exos increased the vessel density and trabecular bone integrity of the femoral head in the GC-induced ONFH rat model, which inhibited the progress of ONFH. CONCLUSIONS: MiR-26a-CD34+-Exos protect the femoral head from damage caused by GCs by strengthening angiogenesis and osteogenesis. The biological effect of miR-26a-CD34+-Exos make them suitable for application in the prevention of GC-induced ONFH.

Abnormal DNA Methylation Induced by Hyperglycemia Reduces CXCR 4 Gene Expression in CD 34+ Stem Cells.

Background CD 34+ stem/progenitor cells are involved in vascular homeostasis and in neovascularization of ischemic tissues. The number of circulating CD 34+ stem cells is a predictive biomarker of adverse cardiovascular outcomes in diabetic patients. Here, we provide evidence that hyperglycemia can be "memorized" by the stem cells through epigenetic changes that contribute to onset and maintenance of their dysfunction in diabetes mellitus. Methods and Results Cord-blood-derived CD 34+ stem cells exposed to high glucose displayed increased reactive oxygen species production, overexpression of p66shc gene, and downregulation of antioxidant genes catalase and manganese superoxide dismutase when compared with normoglycemic cells. This altered oxidative state was associated with impaired migration ability toward stromal-cell-derived factor 1 alpha and reduced protein and mRNA expression of the C-X-C chemokine receptor type 4 ( CXCR 4) receptor. The methylation analysis by bisulfite Sanger sequencing of the CXCR 4 promoter revealed a significant increase in DNA methylation density in high-glucose CD 34+ stem cells that negatively correlated with mRNA expression (Pearson r=-0.76; P=0.004). Consistently, we found, by chromatin immunoprecipitation assay, a more transcriptionally inactive chromatin conformation and reduced RNA polymerase II engagement on the CXCR 4 promoter. Notably, alteration of CXCR 4 DNA methylation, as well as transcriptional and functional defects, persisted in high-glucose CD 34+ stem cells despite recovery in normoglycemic conditions. Importantly, such an epigenetic modification was thoroughly confirmed in bone marrow CD 34+ stem cells isolated from sternal biopsies of diabetic patients undergoing coronary bypass surgery. Conclusions CD 34+ stem cells "memorize" the hyperglycemic environment in the form of epigenetic modifications that collude to alter CXCR 4 receptor expression and migration.

Development of Fe3O4/Ag core/shell-based multifunctional immunomagnetic nanoparticles for isolation and detection of CD34+ stem cells.

Fe3O4/Ag core/shell nanoparticles functionalized with the free amino (NH2) functional groups (Fe3O4/Ag-NH2) were conjugated with fluorescent electron coupled dye (ECD)-antiCD34 antibody using the 1-ethyl-3-(3'-dimethyl-aminopropyl) carbodiimide (EDC) catalyst (ECD - Electron Coupled Dye or R Phycoerythrin-Texas Red is a fluorescent organic dye attached to the antibody). The characteristic fluorescence of ECD in the antibody was investigated and was used as a good indicator for estimating the percentage of the antibodies that were successfully conjugated with the nanoparticles. The conjugation efficiency was found to increase depending on the VNP:VAB ratio, where VNP and VAB are the volumes of the nanoparticle solution (concentration of 50 ppm) and the as-purchased antibody solution, respectively. The conjugation efficiency rapidly increased from approximately 18% to approximately 70% when VNP:VAB was increased from 2:1 to 100:1, and it gradually reached the saturated state at an efficiency of 95%, as the VNP:VAB was equal to 300:1. The bioactivity of the abovementioned conjugation product (denoted by Fe3O4/Ag-antiCD34) was evaluated in an experiment for the collection of stem cells from bone marrow samples.

Transendocardial CD34+ Cell Transplantation in Noncompaction Cardiomyopathy: First-in-Man Case Study.

Noncompaction cardiomyopathy is a rare congenital heart disorder characterized by an arrest of the myocardial compaction process. This results in the altered formation of coronary microvessels with a resulting decrease in myocardial perfusion. Transendocardial CD34+ cell transplantation has been shown to increase myocardial perfusion and function in patients with non-ischemic heart failure. In our first-in-man case study, we investigated the feasibility, safety and clinical effect of transendocardial CD34+ cell therapy in a patient with noncompaction cardiomyopathy.

Expression of Intracellular Reactive Oxygen Species in Hematopoietic Stem Cells Correlates with Time to Neutrophil and Platelet Engraftment in Patients Undergoing Autologous Bone Marrow Transplantation.

Reactive oxygen species (ROS) play important roles in hematopoiesis and regulate the self-renewal, migration, and myeloid differentiation of hematopoietic stem cells (HSCs). This study was conducted to determine whether ROS levels in donor HSCs correlate with neutrophil and platelet engraftment in patients after bone marrow transplantation. Cryopreserved HSC samples from 51 patients who underwent autologous transplantation were studied. Levels of intracellular ROS were assessed by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) in the CD45+/CD34+ HSC population. Colony forming unit assays were performed on HSCs isolated from the ROShigh and ROSlow populations to assess the differentiation potential of these 2 cell subsets. Distinct populations of ROShigh and ROSlow cells were evident in all patient samples. The median percentage of ROShigh expressing HSCs in the study cohort was 75.8% (range, 2% to 95.2%). A significant correlation was identified between the percentage of ROShigh stem cells present in the hematopoietic progenitor cells collected by apheresis product infused and the time to neutrophil engraftment (P < .001, r = -.54), as well as time to plt20, plt50, and plt100 (P < 0.001; r = -.55, -.59, and -.56 respectively). The dose of CD34+/ROShigh/kg infused also inversely correlated with a shorter time to neutrophil engraftment; time to engraftment for patients receiving > or ≤3 × 106 cells/kg was 11.5 days (range, 9 to 23) versus 14 days (range, 10 to 28), respectively (P = .02). The dose of ROShigh HSCs delivered did not correlate with platelet engraftment. Collectively, these data suggest that the dose of ROShigh stem cells delivered to patients may predict time to neutrophil engraftment after autologous transplantation.

A subset of mobilized human hematopoietic stem cells express germ layer lineage genes which can be modulated by culture conditions.

BACKGROUND: Adult bone marrow contains stem cells that replenish the myeloid and lymphoid lineages. A subset of human and mouse CD34+ bone marrow stem cells can be propagated in culture to autonomously express embryonic stem cell genes and embryonic germ layer lineage genes. The current study was undertaken to determine whether these CD34+ stem cells could be obtained from human blood, whether gene expression could be modulated by culture conditions and whether the cells produce insulin. METHODS: Human peripheral blood buffy coat cells and mobilized CD34+ cells from human blood and from blood from C57Bl/6 J mice were cultured in hybridoma medium or neural stem cell induction medium supplemented with interleukin (IL)-3, IL-6, and stem cell factor (SCF). Changes in mRNA and protein expression were assessed by Western blot analysis and by immunohistochemistry. Mass spectrometry was used to assess insulin production. RESULTS: We were able to culture CD34+ cells expressing embryonic stem cell and embryonic germ layer lineage genes from adult human peripheral blood after standard mobilization procedures and from mouse peripheral blood. Gene expression could be modulated by culture conditions, and the cells produced insulin in culture. CONCLUSION: These results suggest a practical method for obtaining large numbers of CD34+ cells from humans to allow studies on their potential to differentiate into other cell types.