CD34 expression does not correlate with immunophenotypic stem cell or progenitor content in human cord blood products.
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ABSTRACT:
SUBMITTER: Mantri S
PROVIDER: S-EPMC7656928 | biostudies-literature | 2020 Nov
REPOSITORIES: biostudies-literature
Publications
CD34 expression does not correlate with immunophenotypic stem cell or progenitor content in human cord blood products.
Mantri Sruthi S Reinisch Andreas A Dejene Beruh T BT Lyell Deirdre J DJ DiGiusto David L DL Agarwal-Hashmi Rajni R Majeti Ravindra R Weinberg Kenneth I KI Porteus Matthew H MH
Blood advances 20201101 21
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Project description:Background We investigated the effect of human serum albumin (HSA) on human umbilical cord blood (UCB) CD34+ hematopoietic stem/progenitor cells (HSPCs) cultured in vitro and transplanted in vivo. Methods Human umbilical cord blood mononuclear cells were obtained by density gradient centrifugation. CD34+ cells were then sorted by CD34 conjugated magnetic microbeads. The sorted cells were cultured with or without HSA for 8 days in vitro. After 8 days, all cells were harvested for flow phenotyping and colony formation cell (CFC) experiments. The cells were injected into immunodeficient mice (NOD/Shi-scid/IL2Rγnull, NOG) via intravenous injections. From 4 weeks post-transplantation, flow cytometry was used to calculate human cell chimerism in the peripheral blood (PB) every 2 weeks. Flow phenotyping of human cell chimerism in bone marrow and spleen was calculated 16 weeks post-transplantation. Results Compared to the control group, CD34+ cells cultured with HSA increased significantly in vitro. The long-term engraftment of HSPCs and the hematopoietic multilineage reconstruction capacity were preserved by HSA. Normal engraftment of human cells could be maintained via HSA treatment could maintain normal engraftment of human cells in recipient PB. Conclusions Here, we found that HSA was beneficial to maintaining CD34+ cell expansion and short-term colony formation in vitro and optimizing multilineage reconstitution in immunodeficient mice in vivo.
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Project description:BackgroundCurrently, a constant shortage in the supply of platelets has become an important medical and society challenge, especially in developing country, and the in vitro production of megakaryocytic progenitor cells (MPs) from cord blood could represent an effective platelet substitute. In the present study, our objective was to determine the safety and feasibility of ex vivo generated MPs in patients.Methods and findingsMPs were produced and characterized from cord blood mononuclear cells under a serum free medium with cytokines. We investigated the feasibility of expansion and infusion of cord blood-derived MPs in 24 patients with advanced hematological malignancies. The primary end point was the safety and tolerability of the infusion of cord blood-derived MPs. No adverse effects were observed in patients who received ex vivo-generated cells at concentrations of up to a median value of 5.45 × 10(6)cells/kg of body weight. With one year follow-up, acute and chronic GVHD had not been observed among patients who received MPs infusion, even without ABO blood group and HLA typing matching.ConclusionsThese initial results in patients are very encouraging. They suggest that infusion of cord blood-derived MPs appears safe and feasible for treatment of thrombocytopenia.
| S-EPMC3563646 | biostudies-literature
Project description:Umbilical cord blood (UCB) is an advantageous source for hematopoietic stem/progenitor cell (HSPC) transplantation, yet the current strategies for large-scale and cost-effective UCB-HSPC preparation are still unavailable. To overcome these obstacles, we systematically evaluate the feasibility of our newly identified CH02 peptide for ex vivo expansion of CD34 + UCB-HSPCs. We herein report that the CH02 peptide is specifically enriched in HSPC proliferation via activating the FLT3 signaling. Notably, the CH02-based cocktails are adequate for boosting 12-fold ex vivo expansion of UCB-HSPCs. Meanwhile, CH02-preconditioned UCB-HSPCs manifest preferable efficacy upon wound healing in diabetic mice via bidirectional orchestration of proinflammatory and anti-inflammatory factors. Together, our data indicate the advantages of the CH02-based strategy for ex vivo expansion of CD34 + UCB-HSPCs, which will provide new strategies for further development of large-scale HSPC preparation for clinical purposes.
| S-EPMC10577473 | biostudies-literature
Project description:Imatinib mesylate (IM) induces clinical remissions in chronic-phase chronic myeloid leukemia (CML) patients but IM resistance remains a problem. We recently identified several features of CML CD34(+) stem/progenitor cells expected to confer resistance to BCR-ABL-targeted therapeutics. From a study of 25 initially chronic-phase patients, we now demonstrate that some, but not all, of these parameters correlate with subsequent clinical response to IM therapy. CD34(+) cells from the 14 IM nonresponders demonstrated greater resistance to IM than the 11 IM responders in colony-forming cell assays in vitro (P < .001) and direct sequencing of cloned transcripts from CD34(+) cells further revealed a higher incidence of BCR-ABL kinase domain mutations in the IM nonresponders (10%-40% vs 0%-20% in IM responders, P < .003). In contrast, CD34(+) cells from IM nonresponders and IM responders were not distinguished by differences in BCR-ABL or transporter gene expression. Interestingly, one BCR-ABL mutation (V304D), predicted to destabilize the interaction between p210(BCR-ABL) and IM, was detectable in 14 of 20 patients. T315I mutant CD34(+) cells found before IM treatment in 2 of 20 patients examined were preferentially amplified after IM treatment. Thus, 2 properties of pretreatment CML stem/progenitor cells correlate with subsequent response to IM therapy. Prospective assessment of these properties may allow improved patient management.
| S-EPMC2951856 | biostudies-literature
Project description:Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non-enriched mononucleated cells (MNC) using novel azole-based small molecules. Freshly-thawed UCB-MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure-activity-relationship studies of SB203580, a known p38-MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8-fold increase of absolute viable CD45+ CD34+ CD38- CD45RA- progenitors which was at least 3.7-fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600-fold expansion of CD34+ /CD90+ /CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+ CD34+ cells in the PB and BM by day 21 compared to non-expanded and cytokine expanded grafts. The C7 expanded grafts maintained long-term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376-393.
| S-EPMC5905230 | biostudies-literature
Project description:For almost two decades, clinicians have overlooked the diagnostic potential of CD34neg hematopoietic stem cells because of their limited homing capacity relative to CD34posHSCs when injected intravenously. This has contributed to the lack of appeal of using umbilical cord blood in HSC transplantation because its stem cell count is lower than bone marrow. The present study reveals that the homing and engraftment of CD34negHSCs can be improved by adding the Sialyl Lewis X molecule via α1,3-fucosylation. This unlocks the potential for using this more primitive stem cell to treat blood disorders because our findings show CD34negHSCs have the capacity to regenerate cells in the bone marrow of mice for several months. Furthermore, our RNA sequencing analysis revealed that CD34negHSCs have unique adhesion pathways, downregulated in CD34posHSCs, that facilitate interaction with the bone marrow niche. Our findings suggest that CD34neg cells will best thrive when the HSC resides in its microenvironment.
| S-EPMC10845921 | biostudies-literature
Project description:We have analyzed the pattern of gene expression in human primary CD34(+) stem/progenitor cells. We identified 42,399 unique serial analysis of gene expression (SAGE) tags among 106,021 SAGE tags collected from 2.5 x 10(6) CD34(+) cells purified from bone marrow. Of these unique SAGE tags, 21,546 matched known expressed sequences, including 3,687 known genes, and 20,854 were novel without a match. The SAGE tags that matched known sequences tended to be at higher levels, whereas the novel SAGE tags tended to be at lower levels. By using the generation of longer sequences from SAGE tags for gene identification (GLGI) method, we identified the correct gene for 385 of 440 high-copy SAGE tags that matched multiple genes and we generated 198 novel 3' expressed sequence tags from 138 high-copy novel SAGE tags. We observed that many different SAGE tags were derived from the same genes, reflecting the high heterogeneity of the 3' untranslated region in the expressed genes. We compared the quantitative relationship for genes known to be important in hematopoiesis. The qualitative identification and quantitative measure for each known gene, expressed sequence tag, and novel SAGE tag provide a base for studying normal gene expression in hematopoietic stem/progenitor cells and for studying abnormal gene expression in hematopoietic diseases.
| S-EPMC61150 | biostudies-literature
Project description:Background/Objectives: We aimed to identify the molecular signatures of primitive CD34+ and CD34- hematopoietic stem/progenitor cell (HSC/HPC) subsets in cord blood and bone marrow samples. Methods: CD34+ and CD34- HSC/HPC subsets from cord blood and bone marrow were characterized using flow cytometry, real-time PCR, and proteomic analysis to evaluate their phenotypic and molecular profiles. Results: Our findings revealed a significantly higher percentage of Lin-CD34-CD38Low/- (-/-) cells than of Lin-CD34+CD38Low/- (+/-) cells in cord blood. Aldehyde dehydrogenase levels were significantly lower in (-/-) than in (+/-) cells. Clonogenic ability was lower in (-/-) than in (+/-) cells. However, CD34- cells exhibited potent megakaryocyte/erythrocyte differentiation ability. Importantly, the HSC/HPC subsets expressed pluripotency or stemness genes (SOX2, Nanog, and OCT4); however, OCT4 expression significantly increased in (-/-) compared with (+/-) cells. We identified 304 proteins in the HSC/HPC subsets-85.6% had similar expression patterns in the two subsets; only 14.4% were differentially expressed between (-/-) and (+/-) cells. This implies their comparability at the protein level. Certain proteins were implicated in cellular-development-, gene-expression-, and embryonic-development-related signaling networks. Conclusions: Distinct biological and functional characteristics were observed between (-/-) and (+/-) HSC/HPC subsets. Some of the identified proteins may be novel HSC/HPC subsets markers for clinical applications after validation.
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Project description:Unlabelled: Cellular reprogramming or conversion is a promising strategy to generate desired stem cell types from somatic cells. Neural stem cells (NSCs) have the potential to regenerate central nervous system tissue and repair damage in response to injury. However, NSCs are difficult to isolate from human tissues and expand in sufficient quantities for therapy. Here, we report a method to generate neural stem cells from cord blood CD34-positive cells by ectopic expression of OCT4 in a feeder-free system. The induced cells (iNSCs) show a characteristic NSC-like morphology and can be expanded in vitro for more than 20 passages. In addition, the iNSCs are positive for neural stem cell-specific markers such as Nestin and Musashi-1 and are similar in gene expression patterns to a human neural stem cell line. The iNSCs express distinct transcriptional factors for forebrain, hindbrain, and spinal cord regions. Upon differentiation, the iNSCs are able to commit into multilineage mature neural cells. Following in vivo introduction into NOD/SCID mice, iNSCs can survive and differentiate in the mouse brain 3 months post-transplantation. Alternatively, we were also able to derive iNSCs with an episomal vector expressing OCT4. Our results suggest a novel, efficient approach to generate neural precursor cells that can be potentially used in drug discovery or regenerative medicine for neurological disease and injury.SignificanceThis study describes a novel method to generate expandable induced neural stem cells from human cord blood cells in a feeder-free system by a single factor, OCT4. The data are promising for future applications that require the generation of large amounts of autologous neural stem cells in disease modeling and regenerative medicine.
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Project description:Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU) assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP), PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15) or pro-angiogenic (galectin-3) properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP) was the most up-regulated gene.
| S-EPMC3155543 | biostudies-literature