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Background: Cell adhesion molecules (CAMs) play a vital role in cell-cell interactions, immune response modulation, and tumor cell migration. However, the unique role of CAMs in gastric cancer (GC) remains largely unexplored., Methods: This study characterized the genetic alterations and mRNA expression of CAMs. The role of CD34, a representative molecule, was validated in 375 GC tissues. The activity of the CAM pathway was further tested using single-cell and bulk characterization. Next, data from 839 patients with GC from three cohorts was analyzed using univariate Cox and random survival forest methods to develop and validate a CAM-related prognostic model., Results: Most CAM-related genes exhibited multi-omics alterations and were associated with clinical outcomes. There was a strong correlation between increased CD34 expression and advanced clinical staging (P = 0.026), extensive vascular infiltration (P = 0.003), and unfavorable prognosis (Log-rank P = 0.022). CD34 expression was also found to be associated with postoperative chemotherapy and tumor immunotherapy response. Furthermore, the CAM pathway was significantly activated and mediated poor prognosis. Additionally, eight prognostic signature genes (PSGs) were identified in the training cohort. There was a substantial upregulation of the expression of immune checkpoints and a pronounced infiltration of immune cells in GC tissues with high PSG score, which is consistent with the prediction of increased sensitivity to immunotherapy. Moreover, 9 compounds from the CTRPv2 database and 13 from the Profiling Relative Inhibition Simultaneously in Mixture (PRISM) database were identified as potential therapeutic drugs for patients with GC with high PSG score., Conclusion: Thorough understanding of CAM pathways regulation and the innovative PSG score model hold significant implications for medical diagnosis, potentially enhancing personalized treatment strategies and improving patient outcomes in GC management., Competing Interests: Declaration of competing interest None declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Erythroid and myeloid differentiation disorders are commonly occurred in leukemia. Given that the relationship between erythroid and myeloid lineages is still unclear. To find the co-regulators in erythroid and myeloid differentiation might help to find new target for therapy of myeloid leukemia. In hematopoiesis, ALA (alpha lipoic acid) is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors via splicing factor SF3B1. However, further exploration is needed to determine whether ELK1 is a common regulatory factor for erythroid and myeloid differentiation., Methods: In vitro culture of isolated CD34 + , CMPs (common myeloid progenitors) and CD34 + CD371 - HSPCs (hematopoietic stem progenitor cells) were performed to assay the differentiation potential of monocytes, neutrophils, and erythrocytes. Overexpression lentivirus of long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 transduced CD34 + HSPCs were transplanted into NSG mice to assay the human lymphocyte and myeloid differentiation differences 3 months after transplantation. Knocking down of SRSF11, which was high expressed in CD371 + GMPs (granulocyte-monocyte progenitors), upregulated by ALA and binding to ELK1-RNA splicing site, was performed to analyze the function in erythroid differentiation derived from CD34 + CD123 mid CD38 + CD371 - HPCs (hematopoietic progenitor cells). RNA sequencing of L-ELK1 and S-ELK1 overexpressed CD34 + CD123 mid CD38 + CD371 - HPCs were performed to assay the signals changed by ELK1., Results: Here, we presented new evidence that ALA promoted erythroid differentiation by targeting the transcription factor ELK1 in CD34 + CD371 - hematopoietic stem progenitor cells (HSPCs). Overexpression of either the long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 inhibited erythroid-cell differentiation, but knockdown of ELK1 did not affect erythroid-cell differentiation. RNAseq analysis of CD34 + CD123 mid CD38 + CD371 - HPCs showed that L-ELK1 upregulated the expression of genes related to neutrophil activity, phosphorylation, and hypoxia signals, while S-ELK1 mainly regulated hypoxia-related signals. However, most of the genes that were upregulated by L-ELK1 were only moderately upregulated by S-ELK1, which might be due to a lack of serum response factor interaction and regulation domains in S-ELK1 compared to L-ELK1. In summary, the differentiation of neutrophils and erythrocytes might need to rely on the dose of L-ELK1 and S-ELK1 to achieve precise regulation via RNA splicing signals at early lineage commitment., Conclusions: ALA and ELK1 are found to regulate both human granulopoiesis and erythropoiesis via RNA spliceosome, and ALA-ELK1 signal might be the target of human leukemia therapy., (© 2024. The Author(s).)

Replacing a faulty gene with a correct copy has become a viable therapeutic option as a result of recent progress in gene editing protocols. Targeted integration of therapeutic genes in hematopoietic stem cells has been achieved for multiple genes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system and Adeno-Associated Virus (AAV) to carry a donor template. Although this is a promising strategy to correct genetic blood disorders, it is associated with toxicity and loss of function in CD34 + hematopoietic stem and progenitor cells, which has hampered clinical application. Balancing the maximum achievable correction against deleterious effects on the cells is critical. However, multiple factors are known to contribute, and the optimization process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach for optimization of gene correction. Using this approach, we could rapidly investigate and select editing conditions for CD34 + cells with the best possible balance between correction and cell/colony-forming unit (CFU) loss in a parsimonious one-shot experiment. This method revealed that using relatively low doses of AAV2/6 and CRISPR/Cas9 ribonucleoprotein complex, we can preserve the fitness of CD34 + cells and, at the same time, achieve high levels of targeted gene insertion. We then used these optimized editing conditions for the correction of p67 phox -deficient chronic granulomatous disease (CGD), an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections and achieved rescue of p67 phox expression and functional correction of CD34 + -derived neutrophils from a CGD patient.

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.

Hematopoietic stem cell (HSC) gene therapy is currently performed on CD34 + hematopoietic stem and progenitor cells containing less than 1% true HSCs and requiring a highly specialized infrastructure for cell manufacturing and transplantation. We have previously identified the CD34 + CD90 + subset to be exclusively responsible for short- and long-term engraftment. However, purification and enrichment of this subset is laborious and expensive. HSC-specific delivery agents for the direct modification of rare HSCs are currently lacking. Here, we developed novel targeted viral vectors to specifically transduce CD90-expressing HSCs. Anti-CD90 single chain variable fragments (scFvs) were engineered onto measles- and VSV-G-pseudotyped lentiviral vectors that were knocked out for native targeting. We further developed a custom hydrodynamic titration methodology to assess the loading of surface-engineered capsids, measure antigen recognition of the scFv, and predict the performance on cells. Engineered vectors formed with minimal impairment in the functional titer, maintained their ability to fuse with the target cells, and showed highly specific recognition of CD90 on cells ex vivo. Most important, targeted vectors selectively transduced human HSCs with secondary colony-forming potential. Our novel HSC-targeted viral vectors have the potential to significantly enhance the feasibility of ex vivo gene therapy and pave the way for future in vivo applications., Competing Interests: Declaration of interests S.R. is consultant to Forty-Seven Inc. (Gilead Sciences) and Ensoma Inc. H.P.K is or was a consultant to and has or had ownership interests with Rocket Pharmaceuticals, Homology Medicines, VOR Biopharma, and Ensoma Inc. H.P.K. has also been a consultant to CSL Behring and Magenta Therapeutics., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Human cytomegalovirus (HCMV) is a beta herpesvirus that persists indefinitely in the human host through a latent infection. The polycistronic UL133-UL138 gene locus of HCMV encodes genes regulating latency and reactivation. While UL138 is pro-latency, restricting virus replication in CD34 + hematopoietic progenitor cells (HPCs), UL135 overcomes this restriction and is required for reactivation. By contrast, UL136 is expressed with later kinetics and encodes multiple proteins with differential roles in latency and reactivation. Like UL135 , the largest UL136 isoform, UL136p33, is required for reactivation from latency in HPCs; viruses failing to express either protein are unresponsive to reactivation stimuli. Furthermore, UL136p33 is unstable, and its instability is important for the establishment of latency, and sufficient accumulation of UL136p33 is a checkpoint for reactivation. We hypothesized that stabilizing UL136p33 might overcome the requirement of UL135 for replication. We generated recombinant viruses lacking UL135 that expressed a stabilized variant of UL136p33. Stabilizing UL136p33 did not impact the replication of the UL135 mutant virus in fibroblasts. However, in the context of infection in HPCs, stabilization of UL136p33 strikingly compensated for the loss of UL135, resulting in increased replication in CD34 + HPCs and in humanized NOD- scid IL2Rγ c null (huNSG) mice. This finding suggests that while UL135 is essential for replication in HPCs, it functions largely at steps preceding the accumulation of UL136p33, and that stabilized expression of UL136p33 largely overcomes the requirement for UL135 . Taken together, our genetic evidence indicates an epistatic relationship between UL136p33 and UL135 , whereby UL135 may initiate events early in reactivation that drive the accumulation of UL136p33 to a threshold required for productive reactivation. IMPORTANCE Human cytomegalovirus (HCMV) is one of nine human herpesviruses and a significant human pathogen. While HCMV establishes a lifelong latent infection that is typically asymptomatic in healthy individuals, its reactivation from latency can have devastating consequences in the immunocompromised. Defining viral genes important in the establishment of or reactivation from latency is important to defining the molecular basis of latent and replicative states and in controlling infection and CMV disease. Here we define a genetic relationship between two viral genes in controlling virus reactivation from latency using primary human hematopoietic progenitor cells and humanized mouse models., Competing Interests: The authors declare no conflict of interest.

Mast cells (MCs) are tissue-resident innate immune cells that express the high-affinity receptor for immunoglobulin E and are responsible for host defense and an array of diseases related to immune system. We aimed in this study to characterize the pathways and gene signatures of human cord blood-derived MCs (hCBMCs) in comparison to cells originating from CD34 - progenitors using next-generation knowledge discovery methods. CD34 + cells were isolated from human umbilical cord blood using magnetic activated cell sorting and differentiated into MCs with rhIL-6 and rhSCF supplementation for 6-8 weeks. The purity of hCBMCs was analyzed by flow cytometry exhibiting the surface markers CD117 + CD34 - CD45 - CD23 - FcεR1α dim . Total RNA from hCBMCs and CD34 - cells were isolated and hybridized using microarray. Differentially expressed genes were analyzed using iPathway Guide and Pre-Ranked Gene Set Enrichment Analysis. Next-generation knowledge discovery platforms revealed MC-specific gene signatures and molecular pathways enriched in hCBMCs and pertain the immunological response repertoire., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023. Published by Elsevier Inc.)

Objective: This study aimed to explore the expression of the C-type lectin domain family 4 member G (CLEC4G) gene in the liver sinusoidal endothelial cells (LSECs) during liver pathogenesis, and to evaluate its correlation with CD34 and clinical significance in hepatocellular carcinoma patients., Methods: We conducted bioinformatics analysis of the differential expression of CLEC4G in various human organs, carcinomatous and adjacent tissues. Then, mRNA and protein expression levels of CD34 in hepatocellular carcinoma (HCC) samples were detected via real-time quantitative reverse transcription PCR (qRT-PCR) and immunohistochemical (IHC), respectively. ELISA was applied to detect serum levels of CLEC4G in healthy controls, liver fibrosis and HCC patients., Results: The expressions of mRNA and protein levels of CLEC4G were higher in normal liver tissues, moderately expressed in cirrhotic and para-cancerous tissues ( P <0.001), and lowest in HCC tissues ( P <0.001). We also found high CD34 expression in tumors, which was negatively correlated with CLEC4G at both mRNA and protein levels. Compared to the healthy controls, the CLEC4G levels in liver fibrosis patients and HCC patients gradually became lower ( P <0.001)., Conclusions: The low expression of CLEC4G is potentially correlated with LSEC capillarization and the appearance of micro-vessels. Such a phenomenon may serve as a reliable diagnostic marker for hepatocellular carcinoma., (© 2023 by the Association of Clinical Scientists, Inc.)

Background: CD34 + cells have been used to treat the patients with heart failure, but the outcome is variable. It is of great significance to scrutinize the fate and the mechanism of CD34 + cell differentiation in vivo during heart failure and explore its intervention strategy., Methods: We performed single-cell RNA sequencing (scRNA-seq) of the total non-cardiomyocytes and enriched Cd34-tdTomato + lineage cells in the murine (male Cd34-CreERT2; Rosa26-tdTomato mice) pressure overload model (transverse aortic constriction, TAC), and total non-cardiomyocytes from human adult hearts. Then, in order to determine the origin of CD34 + cell that plays a role in myocardial fibrosis, bone marrow transplantation model was performed. Furthermore, to further clarify the role of CD34 + cells in myocardial remodeling in response to TAC injury, we generated Cd34-CreERT2; Rosa26-eGFP-DTA (Cre/DTA) mice., Results: By analyzing the transcriptomes of 59,505 single cells from the mouse heart and 22,537 single cells from the human heart, we illustrated the dynamics of cell landscape during the progression of heart hypertrophy, including CD34 + cells, fibroblasts, endothelial and immune cells. By combining genetic lineage tracing and bone marrow transplantation models, we demonstrated that non-bone-marrow-derived CD34 + cells give rise to fibroblasts and endothelial cells, while bone-marrow-derived CD34 + cell turned into immune cells only in response to pressure overload. Interestingly, partial depletion of CD34 + cells alleviated the severity of myocardial fibrosis with a significant improvement of cardiac function in Cd34-CreERT2; Rosa26-eGFP-DTA model. Similar changes of non-cardiomyocyte composition and cellular heterogeneity of heart failure were also observed in human patient with heart failure. Furthermore, immunostaining showed a double labeling of CD34 and fibroblast markers in human heart tissue. Mechanistically, our single-cell pseudotime analysis of scRNA-seq data and in vitro cell culture study revealed that Wnt-β-catenin and TGFβ1/Smad pathways are critical in regulating CD34 + cell differentiation toward fibroblasts., Conclusions: Our study provides a cellular landscape of CD34 + cell-derived cells in the hypertrophy heart of human and animal models, indicating that non-bone-marrow-derived CD34 + cells differentiating into fibroblasts largely account for cardiac fibrosis. These findings may provide novel insights for the pathogenesis of cardiac fibrosis and have further potential therapeutic implications for the heart failure., (© 2023. The Author(s).)

Solitary fibrous tumors (SFTs) are ubiquitous soft tissue neoplasms known for their protean histology and potentially aggressive behavior. Although most cases are composed of a monotonous proliferation of spindle cells, some tumors show unusual cytologic features. We have studied 13 SFTs that were characterized by a predominant population of round epithelioid cells with abundant eosinophilic cytoplasm and clear cell changes. The tumors occurred in 8 women and 5 men, aged 36 to 80 years (mean=63 y), and were located within the orbit (3), lower extremity (3), retroperitoneum (2), abdominal cavity (2), and superficial soft tissues of the neck, pelvis, and pubis (1 each). The tumors measured from 3.5 to 24.5 cm. Using a risk assessment system, 6 cases were stratified as low-risk tumors; 3 of these showed no evidence of recurrence or metastases from 6 to 18 years, and 1 tumor in the orbit recurred and led to the patient's demise. Five cases were of intermediate risk; clinical follow-up showed no evidence of recurrence or metastases from 3 to 4 years in 3 patients, and 1 patient suffered a recurrence 4 years after diagnosis. Two cases were high risk; 1 patient died after 1 year and the second patient experienced local recurrence at 4 years. Immunohistochemical studies showed nuclear positivity for STAT6 in 10 cases. CD34 immunohistochemistry was positive in 11 cases. A NAB2::STAT6 rearrangement was present in all cases. Epithelioid and clear cell SFT should be considered in the differential diagnosis of soft tissue neoplasms with epithelioid and clear cell morphology., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

In β-thalassemia, either γ-globin induction to form fetal hemoglobin (α2γ2) or β-globin repair to restore adult hemoglobin (α2β2) could be therapeutic. ABE8e, a recently evolved adenine base editor variant, can achieve efficient adenine conversion, yet its application in patient-derived hematopoietic stem cells needs further exploration. Here, we purified ABE8e for ribonucleoprotein electroporation of β-thalassemia patient CD34 + hematopoietic stem and progenitor cells to introduce nucleotide substitutions that upregulate γ-globin expression in the BCL11A enhancer or in the HBG promoter. We observed highly efficient on-target adenine base edits at these two regulatory regions, resulting in robust γ-globin induction. Moreover, we developed ABE8e-SpRY, a near-PAMless ABE variant, and successfully applied ABE8e-SpRY RNP to directly correct HbE and IVS II-654 mutations in patient-derived CD34 + HSPCs. Finally, durable therapeutic editing was produced in self-renewing repopulating human HSCs as assayed in primary and secondary recipients. Together, these results support the potential of ABE-mediated base editing in HSCs to treat inherited monogenic blood disorders., (© 2023. The Author(s).)

The nonhuman primate (NHP) animal model is an important predictive preclinical model for developing gene and cell therapies. It is also an experimental animal model used to study hematopoietic stem and progenitor cell (HSPC) biology, with the capability of serving as a step for the translation of the basic research concepts from small animals to humans. Lentiviral vectors are currently the standard gene delivery vehicles for transduction of HSPCs in the clinical setting. They have proven to be less genotoxic and more efficient than the previously used murine γ-retroviruses. Transplantation of lentiviral vector-transduced HSPCs into autologous macaques has been well developed over the past two decades. In this chapter, we provide detailed methodologies for lentiviral vector transduction of rhesus macaque HSPCs, including production and titration of lentiviral vector, purification of CD34 + HSPCs, and lentiviral vector transduction and assessment., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Ex vivo gene therapy procedures targeting hematopoietic stem and progenitor cells (HSPCs) predominantly utilize lentivirus-based vectors for gene transfer. We provide the first pre-clinical evidence of the therapeutic utility of a foamy virus vector (FVV) for the genetic correction of human leukocyte adhesion deficiency type 1 (LAD-1), an inherited primary immunodeficiency resulting from mutation of the β2 integrin common chain, CD18. CD34 + HSPCs isolated from a severely affected LAD-1 patient were transduced under a current good manufacturing practice-compatible protocol with FVV harboring a therapeutic CD18 transgene. LAD-1-associated cellular chemotactic defects were ameliorated in transgene-positive, myeloid-differentiated LAD-1 cells assayed in response to a strong neutrophil chemoattractant in vitro . Xenotransplantation of vector-transduced LAD-1 HSPCs in immunodeficient (NSG) mice resulted in long-term (∼5 months) human cell engraftment within murine bone marrow. Moreover, engrafted LAD-1 myeloid cells displayed in vivo levels of transgene marking previously reported to ameliorate the LAD-1 phenotype in a large animal model of the disease. Vector insertion site analysis revealed a favorable vector integration profile with no overt evidence of genotoxicity. These results coupled with the unique biological features of wild-type foamy virus support the development of FVVs for ex vivo gene therapy of LAD-1.

Pluripotent human embryonic stem cell (hESC) lines are a valuable in vitro tool to differentiate specific cell lineages, including cells from all three germ layers, i.e., neuronal cells, myocytes, and hematopoietic cells, including progenitors (described here), lymphoid cells, and myeloid cells. However, dramatically different cell subtypes and functional properties of specific cells can arise depending on the differentiation technique used. We previously optimized hematopoietic stem cell differentiation from two different NIH-approved hESC lines to generate CD34 + hematopoietic progenitor cells (HPCs). Infection of these HPCs with a common herpesvirus (human cytomegalovirus) results in maintenance of viral latency, capability of viral reactivation, recapitulation of viral mutant phenotypes, and virus-induced myelosuppression of hematopoietic differentiation. However, different HPC subpopulations support different viral latency and reactivation phenotypes, and different hESC-to-HPC differentiation methods alter the ratio of stem cell subsets. In addition, differences in differentiation methods are dependent on both protocol/reagents and user techniques. Here, we report a simplified and optimized method to generate large numbers of CD34 + HPCs with consistent phenotypes and demonstrate a comparison of several common methods that can be used to control the ratio of available HPC subpopulations. A key aspect of this approach is that we achieve consistency in differentiation across users in different laboratories and, importantly, among newly trained individuals. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maintenance of human embryonic stem cells (hESCs) Basic Protocol 2: Differentiation of hESCs to hematopoietic progenitor cells (HPCs) Basic Protocol 3: Downstream functional differentiation of hESC-derived HPCs to mature lineages Support Protocol 1: Freezing and testing frozen batches of hESCs Support Protocol 2: Counting hESCs Support Protocol 3: Phenotyping by flow cytometry., (© 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Myeloblast expansion is a hallmark of disease progression and comprises CD34+ hematopoietic stem and progenitor cells (HSPC). How this compartment evolves during disease progression in chronic myeloid neoplasms is unknown. Using single-cell RNA sequencing and high-parameter flow cytometry, we show that chronic myelomonocytic leukemia (CMML) CD34+ HSPC can be classified into three differentiation trajectories: monocytic, megakaryocyte-erythroid progenitor (MEP), and normal-like. Hallmarks of monocytic-biased trajectory were enrichment of CD120b+ inflammatory granulocyte-macrophage progenitor (GMP)-like cells, activated cytokine receptor signaling, phenotypic hematopoietic stem cell (HSC) depletion, and adverse outcomes. Cytokine receptor diversity was generally an adverse feature and elevated in CD120b+ GMPs. Hypomethylating agents decreased monocytic-biased cells in CMML patients. Given the enrichment of RAS pathway mutations in monocytic-biased cells, NRAS-competitive transplants and LPS-treated xenograft models recapitulated monocytic-biased CMML, suggesting that hematopoietic stress precipitates the monocytic-biased state. Deconvolution of HSPC compartments in other myeloid neoplasms and identifying therapeutic strategies to mitigate the monocytic-biased differentiation trajectory should be explored., Significance: Our findings establish that multiple differentiation states underlie CMML disease progression. These states are negatively augmented by inflammation and positively affected by hypomethylating agents. Furthermore, we identify HSC depletion and expansion of GMP-like cells with increased cytokine receptor diversity as a feature of myeloblast expansion in inflammatory chronic myeloid neoplasms. This article is highlighted in the In This Issue feature, p. 476., (©2022 American Association for Cancer Research.)

Objective: Animal environments for the growth of stem cells cause the transmission of some diseases and immune problems for the recipient. Accordingly, replacing these environments with healthy environments, at least with human resources, is essential.  One of the media that can be used as an alternative to animal serums is Wharton acellular jelly (AWJ).  Therefore, in this study, we intend to replace FBS with Wharton jelly and investigate its effect on the expression of megakaryocyte-related genes and markers in stem cells., Materials and Methods: In this study, cord blood-derived CD34 positive HSCs were cultured and expanded in the presence of cytokines including SCF, TPO, and FLT3-L. Then, the culture of expanded CD34 positive HSCs was performed in two groups: 1) IMDM culture medium containing 10% FBS and 100 ng / ml thrombopoietin cytokine 2) IMDM culture medium containing 10% AWJ, 100 ng / ml thrombopoietin cytokine.  Finally, CD41 expressing cells were analyzed with the flow cytometry method. The genes related to megakaryocyte lineage including FLI1 and GATA2 were also evaluated using the RT-PCR technique.  Results: The expression of CD41, a specific marker of megakaryocyte lineage in culture medium containing Wharton acellular jelly was increased compared to the FBS group. Additionally, the expression of GATA2 and FLI1 genes was significantly increased related to the control group., Conclusion: This study provided evidence of differentiation of CD34 positive hematopoietic stem cells from umbilical cord blood to megakaryocytes in a culture medium containing AWJ., .

Background: The increasing number of clinical trials for induced pluripotent stem cell (iPSC)-derived cell therapy products makes the production on clinical grade iPSC more and more relevant and necessary. Cord blood banks are an ideal source of young, HLA-typed and virus screened starting material to produce HLA-homozygous iPSC lines for wide immune-compatibility allogenic cell therapy approaches. The production of such clinical grade iPSC lines (haplolines) involves particular attention to all steps since donor informed consent, cell procurement and a GMP-compliant cell isolation process., Methods: Homozygous cord blood units were identified and quality verified before recontacting donors for informed consent. CD34+ cells were purified from the mononuclear fraction isolated in a cell processor, by magnetic microbeads labelling and separation columns., Results: We obtained a median recovery of 20.0% of the collected pre-freezing CD34+, with a final product median viability of 99.1% and median purity of 83.5% of the post-thawed purified CD34+ population., Conclusions: Here we describe our own experience, from unit selection and donor reconsenting, in generating a CD34+ cell product as a starting material to produce HLA-homozygous iPSC following a cost-effective and clinical grade-compliant procedure. These CD34+ cells are the basis for the Spanish bank of haplolines envisioned to serve as a source of cell products for clinical research and therapy., (© 2022. The Author(s).)

X-linked Severe Combined Immunodeficiency (SCID-X1) due to IL2RG mutations is potentially fatal in infancy where 'emergency' life-saving stem cell transplant may only achieve incomplete immune reconstitution following transplant. Salvage therapy SCID-X1 patients over 2 years old (NCT01306019) is a non-randomized, open-label, phase I/II clinical trial for administration of lentiviral-transduced autologous hematopoietic stem cells following busulfan (6 mg/kg total) conditioning. The primary and secondary objectives assess efficacy in restoring immunity and safety by vector insertion site analysis (VISA). In this ongoing study (19 patients treated), we report VISA in blood lineages from first eight treated patients with longer follow up found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of the High Mobility Group AT-hook 2 gene. All eight patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but without disturbance of hematopoiesis. Our molecular analysis demonstrated a cryptic splice site within the chicken β-globin hypersensitivity 4 insulator element in the vector generating truncated mRNA transcripts from many transcriptionally active gene containing forward-oriented intronic vector insert. A two base-pair change at the splice site within the lentiviral vector eliminated splicing activity while retaining vector functional capability. This highlights the importance of functional analysis of lentivectors for cryptic splicing for preclinical safety assessment and a redesign of clinical vectors to improve safety., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Monitoring of measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) is predictive of disease recurrence and may identify patients who benefit from treatment intensification. Current MRD techniques rely on multicolor flow cytometry or molecular methods, but are limited in applicability or sensitivity. We evaluated the feasibility of a novel approach for MRD detection in peripheral blood (PB), which combines immunomagnetic preenrichment and fluorescence-activated cell sorting (FACS) for isolation of CD34+ cells with error-reduced targeted next-generation sequencing (NGS). For clinical validation, we retrospectively analyzed 429 PB and 55 bone marrow (BM) samples of 40 patients with AML or high-risk MDS, with/without molecular relapse based on CD34+ donor chimerism (DC), in complete remission after allogeneic stem cell transplantation. Enrichment of CD34+ cells for NGS increased the detection of mutant alleles in PB ∼1000-fold (median variant allele frequency, 1.27% vs 0.0046% in unsorted PB; P < .0001). Although a strong correlation was observed for the parallel analysis of CD34+ PB cells with NGS and DC (r = 0.8601), the combination of FACS and NGS improved sensitivity for MRD detection in dilution experiments ∼10-fold to levels of 10-6. In both assays, MRD detection was superior using PB vs BM for CD34+ enrichment. Importantly, NGS on CD34+ PB cells enabled prediction of molecular relapse with high sensitivity (100%) and specificity (91%), and significantly earlier (median, 48 days; range, 0-281; P = .0011) than by CD34+ DC or NGS of unsorted PB, providing additional time for therapeutic intervention. Moreover, panel sequencing in CD34+ cells allowed for the early assessment of clonal trajectories in hematological complete remission., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Side population (SP) is known to include therapy-resistant cells in various cancers. Here, we analyzed SP using multiple myeloma (MM) samples. The SP accounted for 2.96% in MM cells from newly diagnosed MM (NDMM). CD34 was expressed in 47.8% of SP cells, but only in 2.11% of bulk MM cells. CD34 + MM cells expressed more immature cell surface markers and a gene signature than CD34 - MM cells. CD34 + but not CD34 - MM cells possessed clonogenic activities and showed long-term self-renewal activities in xenotransplantation assays. Similarly, whereas 2.20% of MM cells were CD34 + in NDMM (n = 38), this proportion increased to 42.6% in minimal residual disease (MRD) samples (n = 16) (p < 0.001) and to 17.7% in refractory/relapsed MM (RRMM) (n = 30) (p < 0.01). Cell cycle analysis showed that 24.7% of CD34 + MM cells from NDMM were in G0 phase while this proportion was 54.9% in MRD (p < 0.05) and 14.5% in RRMM, reflecting the expansion of MM. Together, CD34 + MM cells with long-term self-renewal activities persist as MRD in cell cycle quiescence or remain as therapy-resistant cells in RRMM, substantiating the necessity of targeting this population to improve clinical outcomes of MM., (© 2022. Japanese Society of Hematology.)

Background: Coronary endothelial dysfunction (CED) causes angina/ischemia in patients with nonobstructive coronary artery disease (NOCAD). Patients with CED have decreased number and function of CD34+ cells involved in normal vascular repair with microcirculatory regenerative potential and paracrine anti-inflammatory effects. We evaluated safety and potential efficacy of intracoronary autologous CD34+ cell therapy for CED., Methods: Twenty NOCAD patients with invasively diagnosed CED and persistent angina despite maximally tolerated medical therapy underwent baseline exercise stress test, GCSF (granulocyte colony stimulating factor)-mediated CD34+ cell mobilization, leukapheresis, and selective 1×10 5 CD34+ cells/kg infusion into left anterior descending. Invasive CED evaluation and exercise stress test were repeated 6 months after cell infusion. Primary end points were safety and effect of intracoronary autologous CD34+ cell therapy on CED at 6 months of follow-up. Secondary end points were change in Canadian Cardiovascular Society angina class, as-needed sublingual nitroglycerin use/day, Seattle Angina Questionnaire scores, and exercise time at 6 months. Change in CED was compared with that of 51 historic control NOCAD patients treated with maximally tolerated medical therapy alone., Results: Mean age was 52±13 years; 75% were women. No death, myocardial infarction, or stroke occurred. Intracoronary CD34+ cell infusion improved microvascular CED (%acetylcholine-mediated coronary blood flow increased from 7.2 [-18.0 to 32.4] to 57.6 [16.3-98.3]%; P =0.014), decreased Canadian Cardiovascular Society angina class (3.7±0.5 to 1.7±0.9, Wilcoxon signed-rank test, P =0.00018), and sublingual nitroglycerin use/day (1 [0.4-3.5] to 0 [0-1], Wilcoxon signed-rank test, P =0.00047), and improved all Seattle Angina Questionnaire scores with no significant change in exercise time at 6 months of follow-up. Historic control patients had no significant change in CED., Conclusions: A single intracoronary autologous CD34+ cell infusion was safe and may potentially be an effective disease-modifying therapy for microvascular CED in humans. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03471611.

Kinase signaling fuels growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Yet its role in leukemia initiation is unclear and has not been shown in primary human hematopoietic cells. We previously described activating mutations in interleukin-7 receptor alpha (IL7RA) in poor-prognosis "ph-like" BCP-ALL. Here we show that expression of activated mutant IL7RA in human CD34 + hematopoietic stem and progenitor cells induces a preleukemic state in transplanted immunodeficient NOD/LtSz-scid IL2Rγ null mice, characterized by persistence of self-renewing Pro-B cells with non-productive V(D)J gene rearrangements. Preleukemic CD34 + CD10 high CD19 + cells evolve into BCP-ALL with spontaneously acquired Cyclin Dependent Kinase Inhibitor 2 A (CDKN2A) deletions, as commonly observed in primary human BCP-ALL. CRISPR mediated gene silencing of CDKN2A in primary human CD34 + cells transduced with activated IL7RA results in robust development of BCP-ALLs in-vivo. Thus, we demonstrate that constitutive activation of IL7RA can initiate preleukemia in primary human hematopoietic progenitors and cooperates with CDKN2A silencing in progression into BCP-ALL., (© 2022. The Author(s).)

CD34 + CD133 + CD90 + hematopoietic stem cells (HSCs) are responsible for long-term multilineage hematopoiesis, and the high frequency of gene-modified HSCs is crucial for the success of hematopoietic stem and progenitor cell (HSPC) gene therapy. However, the ex vivo culture and gene manipulation steps of HSPC graft preparation significantly reduce the frequency of HSCs, thus necessitating large doses of HSPCs and reagents for the manipulation. In this study, we identified a combination of small molecules, Resveratrol, UM729, and SR1 that preferentially expands CD34 + CD133 + CD90 + HSCs over other subpopulations of adult HSPCs in ex vivo culture. The preferential expansion enriches the HSCs in ex vivo culture, enhances the adhesion, and results in a sixfold increase in the long-term engraftment in NSG mice. Further, the culture-enriched HSCs are more responsive to gene modification by lentiviral transduction and gene editing, increasing the frequency of gene-modified HSCs up to 10-fold in vivo . The yield of gene-modified HSCs obtained by the culture enrichment is similar to the sort-purification of HSCs and superior to Cyclosporin-H treatment. Our study addresses a critical challenge of low frequency of gene modified HSCs in HSPC graft by developing and demonstrating a facile HSPC culture condition that increases the frequency of gene-modified cells in vivo . This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

β-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the β chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent β-thalassemia who were infused with autologous CD34 + cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34 + cells, expression of the transduced β-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34 + cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Poor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34 + human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo., Methods: Human MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34 + cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34 + cells. In addition, CD34 + cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34 + cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice., Results: We did not observe significant differences in viability, cell cycle and ROS expression between CD34 + cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34 + cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34 + cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34 + cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34 + co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively)., Conclusions: Co-transplantation of human CD34 + cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche., (© 2021. The Author(s).)

Myeloproliferative neoplasms (MPN), comprising essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), are hematological disorders of the myeloid lineage characterized by hyperproliferation of mature blood cells. The prediction of the clinical course and progression remains difficult and new therapeutic modalities are required. We conducted a CD34 + gene expression study to identify signatures and potential biomarkers in the different MPN subtypes with the aim to improve treatment and prevent the transformation from the rather benign chronic state to a more malignant aggressive state. We report here on a systematic gene expression analysis (GEA) of CD34 + peripheral blood or bone marrow cells derived from 30 patients with MPN including all subtypes (ET (n = 6), PV (n = 11), PMF (n = 9), secondary MF (SMF; post-ET-/post-PV-MF; n = 4)) and six healthy donors. GEA revealed a variety of differentially regulated genes in the different MPN subtypes vs. controls, with a higher number in PMF/SMF (200/272 genes) than in ET/PV (132/121). PROGENγ analysis revealed significant induction of TNFα/NF-κB signaling (particularly in SMF) and reduction of estrogen signaling (PMF and SMF). Consistently, inflammatory GO terms were enriched in PMF/SMF, whereas RNA splicing-associated biological processes were downregulated in PMF. Differentially regulated genes that might be utilized as diagnostic/prognostic markers were identified, such as AREG, CYBB, DNTT, TIMD4, VCAM1, and S100 family members (S100A4/8/9/10/12). Additionally, 98 genes (including CLEC1B, CMTM5, CXCL8, DACH1, and RADX) were deregulated solely in SMF and may be used to predict progression from early to late stage MPN., (© 2021. The Author(s).)

Thalassemia or sickle cell patients with hereditary persistence of fetal hemoglobin (HbF) have an ameliorated clinical phenotype and, in some cases, can achieve transfusion independence. Inactivation via genome editing of γ-globin developmental suppressors, such as BCL11A or LRF/ZBTB7A, or of their binding sites, have been shown to significantly increase expression of endogenous HbF. To broaden the therapeutic window beyond a single-editing approach, we have explored combinations of cis- and trans-editing targets to enhance HbF reactivation. Multiplex mutagenesis in adult CD34+ cells was well tolerated and did not lead to any detectable defect in the cells' proliferation and differentiation, either in vitro or in vivo. The combination of 1 trans and 1 cis mutation resulted in high editing retention in vivo, coupled with almost pancellular HbF expression in NBSGW mice. The greater in vivo performance of this combination was also recapitulated using a novel helper-dependent adenoviral-CRISPR vector (HD-Ad-dualCRISPR) in CD34+ cells from β-thalassemia patients transplanted to NBSGW mice. A pronounced increase in HbF expression was observed in human red blood cells in mice with established predominant β0/β0-thalassemic hemopoiesis after in vivo injection of the HD-Ad-dualCRISPR vector. Collectively, our data suggest that the combination of cis and trans fetal globin reactivation mutations has the potential to significantly increase HbF both totally and on a per cell basis over single editing and could thus provide significant clinical benefit to patients with severe β-globin phenotype., (© 2021 by The American Society of Hematology.)

[Figure: see text].

Regulation of the IL-5 receptor alpha ( IL5RA ) gene is complicated, with two known promoters (P1 and P2) driving transcription, and two known isoforms (transmembrane and soluble) dichotomously affecting the signaling potential of the protein products. Here, we sought to determine the patterns of P1 and P2 promoter usage and transcription factor occupancy during primary human eosinophil development from CD34 + hematopoietic stem cell progenitors. We found that during eosinophilopoiesis, both promoters were active but subject to distinct temporal regulation, coincident with combinatorial interactions of transcription factors, including GATA-1, PU.1, and C/EBP family members. P1 displayed a relatively constant level of activity throughout eosinophil development, while P2 activity peaked early and waned thereafter. The soluble IL-5Rα mRNA peaked early and showed the greatest magnitude fold-induction, while the signaling-competent transmembrane isoform peaked moderately. Two human eosinophilic cell lines whose relative use of P1 and P2 were similar to eosinophils differentiated in culture were used to functionally test putative transcription factor binding sites. Transcription factor occupancy was then validated in primary cultures by ChIP. We conclude that IL-5-dependent generation of eosinophils from CD34 + precursors involves complex and dynamic activity including both promoters, several interacting transcription factors, and both signaling and antagonistic protein products.

Introduction: Periodontal disease presents a challenge for modern medicine, and research on the use of stem cells as a treatment is currently underway., Material and Methods: The study included 45 patients who were given a thorough physical examination. Additionally, an evaluation of their medical history of the disease, degree of progression of periodontal disease, and the level of CRP in the blood was carried out. Patients were divided into 4 groups: 4 patients were in the first group with no periodontal disease, 8 patients in the second group with a moderate level, 20 patients in the third group with an advanced level, and 13 patients in the fourth group were toothless. For each group, the use of stem cells as a treatment of antibody-labeled CD34+ stem cells, lymphocytes, and leukocytes was conducted., Results: A statistically significant positive correlation was observed in CD34+ stem cells in proportion to lymphocytes in the moderate (0.80), in the advanced (0.75), and in the toothless groups (0.70). The ratio of CD34+ stem cells to leukocytes was statistically significant in the toothless group (0.92) and in the advanced group (0.91). A statistically significant increase was noted in the level of CRP in the previously mentioned groups of patients, and the highest concentration of CD34+ stem cells in the advanced group., Conclusions: The highest concentration of CD34+ cells was observed in the group of patients with advanced periodontal disease.

Malignant epithelioid soft tissue tumors encompass a wide spectrum of lesions. Among them, Epithelioid Malignant Peripheral Nerve Sheath Tumors (MPNST) constitute a distinct subgroup, accounting for <5% of all MPNST. Epithelioid MPNST are infrequently associated with neurofibromatosis type 1, occasionally arise in a schwannoma and show diffuse S100 and CD34 expression, often combined with INI-1 loss. However, the molecular mechanisms underlying the tumorigenesis of epithelioid MPNST remain largely unknown. We describe a case of a 10-year-old girl with an epithelioid malignancy of the orbit. The tumor proved positive for S100, CD34 and SOX10, and, although INI-1 expression was maintained, the overall features suggested the possibility of an epithelioid MPNST, arising in an unusual location. NGS analysis revealed a novel in-frame BRD4-LEUTX fusion gene. LEUTX plays an important role in embryonal genome activation and its expression is mostly suppressed postnatally. We were able to detect increased levels of LEUTX transcript in the tumor, indicating that BRD4-LEUTX fusion leads to LEUTX re-activation. To our knowledge, this fusion has never been reported previously. Whether the current case represents an example of epithelioid MPNST or a distinct tumor entity remains to be determined., (© 2021 Wiley Periodicals LLC.)

Objectives: This study aims to retrospectively assess C-lectin-like molecule 1 (CLL-1) bimodal expression on CD34+ blasts in acute myeloid leukemia (AML) patients (total N = 306) and explore potential CLL-1 bimodal associations with leukemia and patient-specific characteristics., Methods: Flow cytometry assays were performed to assess the deeper immunophenotyping of CLL-1 bimodality. Cytogenetic analysis was performed to characterize the gene mutation on CLL-1-negative subpopulation of CLL-1 bimodal AML samples., Results: The frequency of a bimodal pattern of CLL-1 expression of CD34 + blasts ranged from 8% to 65% in the different cohorts. Bimodal CLL-1 expression was most prevalent in patients with MDS-related AML (P = .011), ELN adverse risk (P = .002), NPM1 wild type (WT, P = .049), FLT3 WT (P = .035), and relatively low percentages of leukemia-associated immunophenotypes (P = .006). Additional immunophenotyping analysis revealed the CLL-1 - subpopulation may consist of pre-B cells, immature myeloblasts, and hematopoietic stem cells. Furthermore, (pre)-leukemic mutations were detected in both CLL-1 + and CLL-1 - subfractions of bimodal samples (N = 3)., Conclusions: C-lectin-like molecule 1 bimodality occurs in about 25% of AML patients and the CLL-1 - cell population still contains malignant cells, hence it may potentially limit the effectiveness of CLL-1-targeted therapies and warrant further investigation., (© 2021 Genentech Inc. European Journal of Haematology published by John Wiley & Sons Ltd.)

Fibroblastic spindle cell tumors are a heterogeneous group of rare soft tissue tumors that are increasingly recognized as associated with a variety of kinase gene fusions. We report two cases of GAB1-ABL1 fusions in spindle cell tumors that histologically overlap with neurotrophic tyrosine receptor kinase (NTRK)-rearranged spindle cell tumors. The first case occurred in a 76-year-old female who had a large deep-seated spindle cell tumor composed of monotonous ovoid to spindle cells in a background of thick stromal collagen bands with prominent hyalinized vessels and inconspicuous mitoses (<1/10 HPF). Immunohistochemical stains showed co-expression of S100 and CD34. A GAB1-ABL1 fusion was detected by whole transcriptome RNA sequencing. The patient had a partial response to imatinib. The second case was previously described as a solitary fibrous tumor, occurring in a 9-year-old female with a cellular spindle cell tumor with patchy CD34 immunoexpression but no expression of S100. Upon clinicopathologic re-review, including anchored multiplex next-generation sequencing, a GAB1-ABL1 fusion was identified. In summary, we report the first two cases of spindle cell tumors with variable expression of CD34 and/or S100, driven by GAB1-ABL1 gene fusions with histologic overlap with NTRK-rearranged spindle cell tumors, suggesting that ABL-fusions may also be oncogenic drivers within this spectrum of tumors. These cases highlight the evolving understanding of fibroblastic spindle cell tumor biology and the utility of sequencing in identifying a targetable alteration., (© 2021 Wiley Periodicals LLC.)

T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and T-lineage cells from CD34 + cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-μbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34 + cells to CD34 + CD7 + CD5 + proT cells to CD3 + αβ T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34 + CD7 + proT cells into immunodeficient mice demonstrates efficient thymic engraftment and functional maturation of peripheral T cells. DL4-μbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources., (© 2021. The Author(s).)

Background: Successful neoadjuvant chemotherapy (NACT) could improve the surgical resection rate and radical curability of patients with cervical cancer, but only a subset of patients benefits. Therefore, identifying predictive biomarkers are urgently needed. The aim of this study was to evaluate the predictive value of CD34 and Bcl-2 in the NACT effectiveness of cervical cancer., Methods: Sixty-seven patients with locally advanced cervical cancer (FIGO stages IB3, IIA2 or IIB) were classified into two groups based on effective (n = 48) and ineffective (n = 19) response to NACT. Immunohistochemistry was employed to identify CD34 and Bcl-2 expression before and after NACT. We analyzed the associations between the pre-NACT expression of these two biomarkers and the response of NACT. The expression of these two biomarkers before and after NACT was also assessed and compared., Results: More patients were CD34 positive expression before NACT in effective group compared to ineffective group (p = 0.005). However, no statistically significant difference in Bcl-2 expression before NACT were found between two groups (p = 0.084). In NACT effective group, the expression of both CD34 and Bcl-2 after NACT are down-regulated (p < 0.001 and p < 0.001, respectively), while there are no statistical differences between the pre- and post-NACT expression of CD34 and Bcl-2 in NACT ineffective group (p = 0.453 and p = 0.317, respectively)., Conclusion: The positive CD34 expression before NACT may serve as a predictive biomarker for NACT of cervical cancer, but the pre-NACT expression of Bcl-2 is not an independent predictor. The down-regulated expression of these two indicators after NACT may indicate effective NACT., (© 2021. The Author(s).)

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP., (© 2020 International Clinical Cytometry Society.)

The cell surface protein CD34 is expressed in various human tissues and cells, including hematopoietic stem cells, vascular endothelial cells, mucosal dendritic cells, mast cells, eosinophils, microglia, fibrocytes, muscle satellite cells, and platelets. There is a lack of data on the expression of CD34 in canine and porcine tissues. Therefore, we designed a series of immunoblotting, immunohistochemistry, and immunofluorescence experiments to observe CD34 expression in murine, canine, and porcine lungs. We used a rabbit antibody (clone EP373Y) to target the conserved human CD34 C-terminal region and validated its immunoreactivity against mouse lung homogenates. The data showed diffuse bronchiolar and alveolar epithelial localization of CD34 protein in normal murine, canine, and porcine lungs. At 9 or 24 h after bacterial endotoxin exposure, murine CD34 protein shifted to specific bronchoalveolar cells with a punctate pattern, as quantified by CD34 fluorescence. Specific porcine bronchoalveolar cells and leukocytes had significant CD34-positive immunostaining after H3N1 influenza infection. Thus, our study provides fundamental data on the expression of CD34 in lungs and validates an antibody for use in further experiments in these animal species., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Acute basophilic leukaemia (ABL) is a rare subtype of acute myeloid leukaemia (AML); therefore, few data are available about its biology. Herein, we analysed two ABL patients using flow cytometry and next-generation sequencing (NGS). Two cell populations were detected by flow cytometry in both patients. In Case no. 1, blasts (CD34 + , CD203c - , CD117 + , CD123dim + ) and basophils (CD34 - , CD203c + , CD117 ± , CD123 + ) were identified, both of which were found by NGS to harbour the 17p deletion and have loss of heterozygosity of TP53. In Case no. 2, blasts (CD33 + , CD34 + , CD123 - ) and basophils (CD33 + , CD34 + , CD123 + ) were identified. NGS detected NPM1 mutations in either blasts or basophils, and TET2 in both. These data suggest an overlap of the mutational landscape of ABL and AML, including TP53 and TET2 mutations. Moreover, additional mutations or epigenetic factors may contribute for the differentiation into basophilic blasts., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by impaired communication skills, ataxia, motor and balance deficits, intellectual disabilities, and seizures. The genetic cause of AS is the neuronal loss of UBE3A expression in the brain. A novel approach, described here, is a stem cell gene therapy which uses lentivector-transduced hematopoietic stem and progenitor cells to deliver functional UBE3A to affected cells. We have demonstrated both the prevention and reversal of AS phenotypes upon transplantation and engraftment of human CD34+ cells transduced with a Ube3a lentivector in a novel immunodeficient Ube3amat-/pat+ IL2rg-/y mouse model of AS. A significant improvement in motor and cognitive behavioral assays as well as normalized delta power measured by electroencephalogram was observed in neonates and adults transplanted with the gene modified cells. Human hematopoietic profiles observed in the lymphoid organs by detection of human immune cells were normal. Expression of UBE3A was detected in the brains of the adult treatment group following immunohistochemical staining illustrating engraftment of the gene-modified cells expressing UBE3A in the brain. As demonstrated with our data, this stem cell gene therapy approach offers a promising treatment strategy for AS, not requiring a critical treatment window., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: To investigate the protective effects of electroacupuncture and LGNHFD on cerebral ischemia-reperfusion injury, and their effects on the expression of cluster of differentiation 34 (CD 34), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) in the peripheral tissue of the cerebral ischemic semi-dark zone in rats. The influence and mechanism of expression., Methods: Healthy male Sprague-Dawley rats were randomly divided into the sham operation group, model group, Traditional Chinese Medicine (TCM) group, electroacupuncture group, and acupuncture group. The cerebral ischemia model was prepared by middle artery occlusion. The electroacupuncture and TCM groups received electroacupuncture at the ""Neiguan (PC6)"", ""Baihui (GV20)"", and ""Renzhong (GV26)"" acupoints from 4 h after modeling, once daily for 14 d. After modeling, the acupuncture and TCM groups were administered LGNHFD once daily for 14 d. After treatment, the infarct volume and regional cerebral blood flow (rCBF) were measured in each group, the expression of CD34 in the surrounding ischemic penumbra was determined using immunohistochemistry, and the expression of angiogenesis-related factors in the ischemic hemisphere was detected by western blot., Results: Compared with the sham operation group, rCBF was significantly decreased (P < 0.01) and cerebral infarction volume was significantly increased (P < 0.01) in the model group at four time points (5 min and 3, 7, and 14 d after modeling). At three postoperative time points (3, 7, and 14 d), the number of CD34+ cells in the model group were significantly increased (P < 0.01), and the relative expression levels of VEGF, bFGF, and CD34+ in the ischemic hemisphere were significantly increased (P < 0.01, P < 0.05). Compared with the model group, the rCBF of the acupuncture group was significantly increased at 3, 7, and 14 d after intervention (P < 0.01), while the rCBF of the electroacupuncture and TCM groups was significantly increased at 7 and 14 d after intervention (P < 0.01). After 3, 7, and 14 d of intervention, infarct volume was significantly reduced (P < 0.01), the number of CD34+ cells was significantly increased (P < 0.01, P < 0.05), and the relative expression levels of VEGF, bFGF, and CD34 in the ischemic hemisphere were significantly increased (P < 0.01, P < 0.05). Compared with the electroacupuncture and TCM groups, the rCBF of the acupuncture group was significantly increased (P < 0.01), the infarct volume was significantly reduced (P < 0.01), and the relative expression levels of VEGF, bFGF, and CD34 were significantly increased (P < 0.05, P < 0.01) after 3, 7, and 14 d of intervention., Conclusion: Acupuncture therapy is superior to simple drug and electroacupuncture therapy for reducing infarct volume, increasing rCBF and CD34 expression in cells, and promoting capillary regeneration in rats with cerebral ischemia. This may be related to the increased expression of VEGF, bFGF, and CD34 - which are related to cerebrovascular neovascularization - in the ischemic hemispheres of such rats.

High-risk neuroblastoma (HR-NB) is branded with hematogenous metastasis, relapses, and dismal long-term survival. Intensification of consolidation therapy with tandem/triple autologous stem cell (SC) rescue (with bone marrow [BM]/peripheral blood [PB] CD34 + selection) after myeloablative chemotherapy has improved long-term survival. However, the benefit is limited by the indication of NB cells in CD34 + PBSCs, CD34 expression in NB cells, and the risk of reinfusing NB cancer stem cells (NB CSCs) that could lead to post-transplant relapse. We investigated the association of CD34 surface expression (92 patients) with NB evolution/clinical outcomes. CD34 gene-level status in NB was assessed through RNA-Seq data mining (18 cohorts, n, 3324). Genetic landscape of CD34-expressing NB CSCs (CD133 + CD34 + ) was compared with CD34 - CSCs (CD133 + CD34 - ). RNA-seq data revealed equivocal association patterns of CD34 expression with patient survival. Our immunohistochemistry data revealed definite, but rare (mean, 0.73%; range 0.00-7.87%; median, 0.20%) CD34 positivity in NB. CD34 + significantly associated with MYCN amplification (p, 0.003), advanced disease stage (p, 0.016), and progressive disease (PD, p < 0.0009) after clinical therapy. A general high-is-worse tendency was observed in patients with relapsed disease. High CD34 + correlated with poor survival in patients with N-MYC-amplified HR-NB. Gene expression analysis of CD34 + -NB CSCs identified significant up (4631) and downmodulation (4678) of genes compared with NB CSCs that lack CD34. IPA recognized the modulation of crucial signaling elements (EMT, stemness maintenance, differentiation, inflammation, clonal expansion, drug resistance, metastasis) that orchestrate NB disease evolution in CD34 +  CSCs compared with CD34 - CSCs. While the function of CD34 in NB evolution requires further in-depth investigation, careful consideration should be exercised for autologous stem cell rescue with CD34 + selection in NB patients. Graphical abstract.

Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34 + cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect-the accumulation of CD34 + cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineage negative Sca-1 + cKit + ) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.

Human cytomegalovirus (HCMV) microRNAs play essential roles in latency and reactivation in CD34 + hematopoietic progenitor cells (HPCs) via regulation of viral and cellular gene expression. In the present study, we show that HCMV miR-US25-1 targets RhoA, a small GTPase required for CD34 + HPC self-renewal, proliferation, and hematopoiesis. Expression of miR-US25-1 impairs signaling through the nonmuscle myosin II light chain, which leads to a block in cytokinesis and an inhibition of proliferation. Moreover, infection with an HCMV mutant lacking miR-US25-1 resulted in increased proliferation of CD34 + HPCs and a decrease in the proportion of genome-containing cells at the end of latency culture. These observations provide a mechanism by which HCMV limits proliferation to maintain latent viral genomes in CD34 + HPCs. IMPORTANCE Each herpesvirus family establishes latency in a unique cell type. Since herpesvirus genomes are maintained as episomes, the virus needs to devise mechanisms to retain the latent genome during cell division. Alphaherpesviruses overcome this obstacle by infecting nondividing neurons, while gammaherpesviruses tether their genome to the host chromosome in dividing B cells. The betaherpesvirus human cytomegalovirus (HCMV) establishes latency in CD34 + hematopoietic progenitor cells (HPCs), but the mechanism used to maintain the viral genome is unknown. In this report, we demonstrate that HCMV miR-US25-1 downregulates expression of RhoA, a key cell cycle regulator, which results in inhibition of CD34 + HPC proliferation by blocking mitosis. Mutation of miR-US25-1 during viral infection results in enhanced cellular proliferation and a decreased frequency of genome-containing CD34 + HPCs. These results reveal a novel mechanism through which HCMV is able to regulate cell division to prevent viral genome loss during proliferation., (Copyright © 2021 Diggins et al.)

Hematopoietic stem cell (HSC) heterogeneity and hierarchy are a current topic of interest, having major implications for clinical HSC transplantation and basic research on human HSCs. It was long believed that the most primitive HSCs in mammals, including mice and humans, were CD34 antigen positive (CD34 + ). However, 2 decades ago, it was reported that murine long-term multilineage reconstituting HSCs were lineage marker negative (Lin - , i.e., c-kit + Sca-1 + CD34 low/- ), known as CD34 low/- KSL cells. In contrast, human CD34 - HSCs, a counterpart of murine CD34 low/- KSL cells, were hard to identify for a long time mainly because of their rarity. We previously identified very primitive human cord blood (CB)-derived CD34 - severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method and proposed the new concept that CD34 - SRCs (HSCs) reside at the apex of the human HSC hierarchy. Through a series of studies, we identified two positive/enrichment markers: CD133 and GPI-80. The combination of these two markers enabled the development of an ultrahigh-resolution purification method for CD34 - as well as CD34 + HSCs and the successful purification of both HSCs at the single-cell level. Cell population purity is a crucial prerequisite for reliable biological and molecular analyses. Clonal analyses of highly purified human CD34 - HSCs have revealed their potent megakaryocyte/erythrocyte differentiation potential. Based on these observations, we propose a revised road map for the commitment of human CB-derived CD34 - HSCs. This review updates the current understanding of the stem cell nature of human CB-derived primitive CD34 - as well as CD34 + HSCs., Competing Interests: Conflict of interest disclosure The author has no conflicts of interest to disclose., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: This study was aimed to explore the application value of modified closed biopsy technique in puncture biopsy of rabbit VX2 transplanted bone tumor model., Methods: VX2 tumor was transplanted into the bilateral tibia of 30 rabbits through the tibial plateau to make the model of VX2 transplanted bone tumor. Seven days after modeling, the proximal tibia biopsy was performed under the guidance of X-ray, and the biopsy specimen was examined pathologically. The left leg was biopsied with modified closed biopsy technique (experimental group), and the right leg was biopsied with hollow needle (control group). After 14 days of modeling, all rabbits were killed after X-ray examination around the puncture hole, and the soft tissue around the puncture hole was taken for pathological examination, and the expression levels of PCNA and CD34 in the tissue extract were detected by enzyme-linked immunosorbent assay (ELISA)., Results: By the end of the experiment, a total of 3 rabbits died, and finally, 27 rabbits were included in the study. Tumor cells were detected in all the intramedullary specimens obtained by puncture biopsy. On the 14th day after modeling, X-ray showed that the occurrence rate of periosteal reaction and extraosseous high-density shadow around the puncture hole was 14.81% (4/27) in the experimental group and 40.74% (11/27) in the control group. The difference was statistically significant (P<0.05). The pathological results of soft tissue around the puncture hole showed that the tumor cell metastasis rate was 29.63% (8/27) in the experimental group and 100% (27/27) in the control group, and the difference was statistically significant (P<0.05). The expression levels of PCNA and CD34 in the experimental group were lower than those in the control group (P < 0.05)., Conclusion: Both the modified closed biopsy technique and needle aspiration biopsy can provide sufficient biopsy tissue for the diagnosis of VX2-transplanted bone tumor in rabbits. At the same time, the improved closed biopsy technique has a certain application value in preventing local metastasis of tumor cells along the puncture channel.

Despite the increasing demand of cellular therapies for dogs, little is known on the differences between adult and fetal adnexa canine mesenchymal stem cells (MSCs), and data on their metabolic features are lacking. The present study aimed at comparing the characteristics of canine adipose tissue (AT) and umbilical cord matrix (UC) MSCs. Moreover, for the first time in the dog, the cellular bioenergetics were investigated by evaluating the two main metabolic pathways (oxidative phosphorylation and glycolysis) of ATP production. Frozen-thawed samples were used for this study. No differences in mean cell proliferation were found (P>0.05). However, while AT-MSCs showed a progressive increase in doubling time over passages, UC-MSCs showed an initial post freezing-thawing latency. No differences in migration, spheroid formation ability, and differentiation potential were found (P>0.05). RT-PCR analysis confirmed the expression of CD90 and CD44, the lack of CD14 and weak expression of CD34, mostly by AT-MSCs. DLA-DRA1 and DLA-DQA1 were weakly expressed only at passage 0 by UC-MSCs, while they were expressed at different passages for AT-MSCs. There was no difference (P>0.05) in total ATP production between cell cultures, but the ratio between the "mitochondrial ATP Production Rate" and the "glycolytic ATP Production Rate" was higher (P<0.05) in AT- than in UC-MSCs. However, in both MSCs types the mitochondrial respiration was the main pathway of ATP production. Mitochondrial respiration and ATP turnover in UC-MSCs were higher (P<0.05) than in AT-MSCs, but both had a 100% coupling efficiency. These features and the possibility of increasing the oxygen consumption by a spare respiratory capacity of four (AT-MSCSs) and two (UC-MSCs) order of magnitude greater than basal respiration, can be taken as indicative of the cell propensity to differentiate. The findings may efficiently contribute to select the most appropriate MSCs, culture and experimental conditions for transplantation experiments in mesenchymal stem cell therapy for companion animals., Competing Interests: The authors have declared that no competing interests exist.

Background: CD34, a pan-selectin binding protein when glycosylated, has been shown to be involved in leukocyte migration to the site of inflammation. However, only one report is available on the expression and role of CD34 in neutrophil recruitment during acute lung inflammation., Methods: We proceeded to study the role of CD34 in lung neutrophil migration using mouse model of endotoxin induced acute lung inflammation and studied over multiple time points, in generic CD34 knock-out (KO) strain., Results: While there was no difference in BAL total or differential leukocyte counts, lung MPO content was lower in LPS exposed KO compared to WT group at 3 h time-point (p = 0.0308). The MPO levels in CD34 KO mice begin to rise at 9 h (p = 0.0021), as opposed to an early 3 h rise in WT mice (p = 0.0001), indicating that KO mice display delays in lung neutrophil recruitment kinetics. KO mice do not loose endotoxin induced lung vascular barrier properties as suggested by lower BAL total protein at 3 h (p = 0.0452) and 24 h (p = 0.0113) time-points. Several pro-inflammatory cytokines and chemokines (TNF-α, IL-1β, KC, MIP-1α, IL-6, IL-10 and IL-12 p70 sub-unit; p < 0.05) had higher levels in WT compared to KO group, at 3 h. Lung immunofluorescence in healthy WT mice reveals CD34 expression in the bronchiolar epithelium, in addition to alveolar septa., Conclusion: Thus, given CD34's pan-selectin affinity, and expression in the bronchiolar epithelium as well as alveolar septa, our study points towards a role of CD34 in lung neutrophil recruitment but not alveolar migration, cytokine expression and lung inflammation.