Avaliação dos efeitos modulatórios da hematopoese no envelhecimento: Papel das células tronco mesenquimais medulares / Evaluation of the modulatory effects of hematopoiesis during aging: Role of bone marrow mesenchymal stem cells

O envelhecimento é um processo fisiológico que traz consigo uma série de alterações no organismo que se estendem até o nível molecular. Diante disto, este é um processo complexo que afeta diversos tecidos, sendo um deles o hematopoético, local onde, através de interações da Célula Tronco Hematopoética (CTH) com o ambiente ao seu redor, incluindo a Célula Tronco Mesenquimal (CTM), ocorre a hematopoese. Embora já sejam descritas na literatura algumas alterações na medula óssea consequentes do envelhecimento, os mecanismos por trás de tais mudanças permanecem elusivas, principalmente no âmbito das interações celulares ocorrentes na medula óssea. Portanto, este trabalho buscou investigar como o envelhecimento afeta a regulação hematopoética no contexto de sua relação com as CTM medulares. Para esta pesquisa, foram utilizados camundongos machos isogênicos da linhagem C57BL/6, dividindoos em grupos conforme sua idade: jovens (3 ­ 5 meses) e idosos (18 ­ 19 meses). Foi realizada a caracterização do modelo através de aspectos físicos como consumo proteico, variação de peso, entre outros, seguido de avaliação bioquímica e hematológica. Adicionalmente, foram coletadas células medulares e, posteriormente, realizado o isolamento das CTMs. Para estudar a relação destas células com a hematopoese, foram realizados ensaios in vitro utilizando a linhagem celular leucêmica C1498 (TIB-49™, ATCC®) mantidas em contato com o sobrenadante das CTMs isoladas. Quanto aos parâmetros bioquímicos, os animais idosos apresentaram menores níveis de albumina, aspartato alanina transferase (ALT) e de triglicerídeos quando comparados aos animais jovens. Contrariamente, os animais idosos apresentaram um maior nível de colesterol. Na avaliação hematológica, foi constatado pelo hemograma que os animais idosos apresentaram valores comparáveis aos animais jovens, todavia, o mielograma mostrou menor celularidade geral, seguido de menor número de células da linhagem eritroide e maior número de precursores granulocíticos. Através da imunofenotipagem, foi revelado um maior número de CTHs e de precursores grânulosmonocíticos na medula de animais idosos quando comparado aos jovens, e uma menor frequência de progenitores linfoides. Na imunofenotipagem de sangue periférico de animais idosos houve uma redução no número de linfócitos B e de eritrócitos, e aumento na população de células natural killers. Na imunofenotipagem de CTMs, o marcador CD73 apresentou menor expressão nos animais idosos. Avaliando o secretoma destas células estromais, foram encontrados no sobrenadante de CTMs de animais idosos aumentos significativos nas concentrações de CXCL12 e SCF e redução de IL-11. No âmbito molecular, as CTMs de animais idosos apresentaram aumento na expressão de Akt1, Nos e Ppar-γ, e redução na expressão de Csf3 e Cdh2. Adicionalmente, quando comparado a ação das CTMs de animais idosos em relação as CTMs de animais jovens, observou-se que CTMs de animais idosos foram capazes de aumentar a expressão de Sox2, Pou5f1 e Nanog e diminuir a expressão de Cdkn1a de células da linhagem C1498. O sobrenadante de CTMs de animais idosos também resultou na maior proliferação e migração de células da linhagem C1498. Portanto, levando em consideração a importância das CTMs sobre a regulação do sistema hematopoético, pode-se concluir que, no envelhecimento, as CTMs criam um ambiente propício para a proliferação celular no qual a manutenção da pluripotência é estimulada, o que pode acarretar em uma desregulação do sítio hematopoético quando habitado por células malignas

Aging is a physiological process in which occurs a series of alterations in an organism that extend to a molecular level. It is a complex process that affects various tissues, one of them being the bone marrow, wherethrough the interactions of the hematopoietic stem cell (CTH) with its surrounding environment, including with the mesenchymal stem cell (CTM), hematopoiesis takes place. Although some aging-associated alterations in the bone marrow can be found described in the literature, the mechanisms behind said changes remain elusive, especially when regarding the cellular interactions present inside the bone marrow. Therefore, this research aimed to investigate how aging affects the regulation of hematopoiesis in the context of its interactions with bone marrow-derived CTMs. For this investigation, male isogenic C57BL/6 mice were used as animal models. These were separated in two groups according to their age: young (3 ­ 5 months) and aged (18 ­ 19 months). The animal models were characterized by their physical properties such as protein intake and weight variation, followed by biochemical and hematological evaluation. Bone marrow cells were obtained and identified through immunophenotyping, thus isolating different cell populations, including the CTMs. To study the relationship between these cells and hematopoiesis, in vitro assays were conducted utilizing the leukemic cell lineage C1498 (TIB-49™, ATCC®) maintained in contact with the supernatant of isolated CTMs. By their biochemical profile, aged mice showed lower levels of albumin, alanine-aspartate transferase (ALT) and triglycerides compared to the young group. In contrast, aged mice had a higher cholesterol level. Hematological evaluation by total blood count showed similar results between the two groups, however, the myelogram revealed that the aged animals had lower cellularity, with less frequent cells from the erythroid lineage, with an increase in granulocytic precursors. Through immunophenotyping, it was also revealed that aged mice have higher numbers of hematopoietic stem cells, while also being noted a reduced population of lymphoid progenitors. An increase in the granulomonocytic progenitors was also found. Immunophenotyping peripheral blood cells of aged mice revealed reduced numbers of B lymphocytes and erythrocytes, and an increased natural killer cell population. Additionally, the cell surface marker CD73 was found to be less expressed in aged mice CTMs. The secretome of these stromal cells obtained from aged mice showed higher levels of CXCL12 and SCF, and lower levels of IL-11when compared to the young counterparts. At a molecular level, CTMs obtained from aged mice expressed more Akt1, Nos and Ppar-γ, while the expression of Csf3 and Cdh2 was reduced. Additionally, when comparing the effects of aged mice CTMs with young mice CTMs, it was observed that the first expressed were capable of increasing the expression of Sox2, Pou5f1 and Nanog, while decreasing Cdkn1a expression in the C1498 cell lineage. The supernatant obtained from aged mice also favored the proliferation and cell migration of the C1498 cell line. Thus, considering the importance that CTMs have over the hematopoietic system, we can conclude that, in aging, CTMs create a special environment which favors cell proliferation and maintenance of pluripotency, which can result in a dysregulation of the hematopoietic tissue when malignant cells are present

Spatial and temporal expression pattern of somatostatin receptor 2 in mouse / 生物工程学报

Somatostatin (SST) is an inhibitory polypeptide hormone that plays an important role in a variety of biological processes. Somatostatin receptor 2 (SSTR2) is the most widely expressed somatostatin receptor. However, the specific cell types expressing Sstr2 in the tissues have not been investigated. In this study, we detected the expression pattern of SSTR2 protein in mouse at different development stages, including the embryonic 15.5 days and the postnatal 1, 7, 15 days as well as 3 and 6 months, by multicolour immunofluorescence analyses. We found that Sstr2 was expressed in some specific cells types of several tissues, including the neuronal cells and astrocytes in the brain, the mesenchymal cells, the hematopoietic cells, the early hematopoietic stem cells, and the B cells in the bone marrow, the macrophages, the type Ⅱ alveolar epithelial cells, and the airway ciliated cells in the lung, the epithelial cells and the neuronal cells in the intestine, the hair follicle cells, the gastric epithelial cells, the hematopoietic stem cells and the nerve fibre in the spleen, and the tubular epithelial cells in the kidney. This study identified the specific cell types expressing Sstr2 in mouse at different developmental stages, providing new insights into the physiological function of SST and SSTR2 in several cell types.

Effect of Recombinant Human Thrombopoietin (rhTPO) on Hematopoietic Reconstitution in Allogeneic Hematopoietic Stem Cell Transplantation Model / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of recombinant human thrombopoietin (rhTPO) on hematopoietic reconstruction in allogeneic hematopoietic stem cell transplantation (allo-HSCT) model.@*METHODS@#The C57BL/6 mice were employed as the donors, and BALB/c mice as recipients. The bone marrow mononuclear cells of the donor mice were extracted and pretreated, which then were injected with 5×106 per mouse through the tail vein of the recipient to establish an allo-HSCT model. The implantation of hematopoietic stem cells in the recipient mice was detected by flow cytometry on the 28th day after transplantation. Next, the successfully modeled recipient mice were randomly divided into experimental group and control group. The rhTPO was injected into mice in the experimental group on the first day after transplantation, while the saline was injected into mice in the control group. Both groups were injected for 14 consecutive days. The peripheral blood and bone marrow hematopoiesis of the two groups were observed on day 1, 3, 7, 14, and 21 after transplantation.@*RESULTS@#The expression rate of H-2Kb in the bone marrow of recipient mice was 43.85% (>20%) on the 28th day after transplantation, which indicated that the recipient mice were successfully chimerized. Meanwhile, counts of PLTs on the day 3, 7, 14, and 21 after transplantation in the experimental group were higher than those in the control group with statistical significances (P<0.05). In addition, hematopoietic function of bone marrow was suppressed in both groups on day 1, 3 and 7 after transplantation, but hematopoietic bone marrow hyperplasia was better in the experimental group than in the control group. On day 14 and 21 after transplantation, the hematopoietic function of bone marrow in the two groups was recovered, and the experimental group showed more obvious than the control group.@*CONCLUSION@#rhTPO can effectively stimulate the production of PLTs and facilitate the recovery of white blood cells and hemoglobin after allo-HSCT, and promote hematopoietic recovery and reconstitution of bone marrow.

Clinical Analysis of Autologous Peripheral Blood Hematopoietic Stem Cells Mobilization and Collection in Lymphoma Patients / 中国实验血液学杂志

OBJECTIVE@#To analyze the factors influencing collection of autologous peripheral blood hematopoietic stem cells in lymphoma patients.@*METHODS@#Clinical data of 74 patients who received autologous peripheral blood hematopoietic stem cells mobilization and collection in the 940th Hospital of Joint Logistic Support Force of PLA from April 2009 to April 2021 were collected. The effects of gender, age, disease type, stage, course of disease, chemotherapy cycle number, relapse, radiotherapy, disease status and blood routine indexes on the day of collection on peripheral blood hematopoietic stem cell collection were analyzed.@*RESULTS@#The success rate of collection was 95.9%(71/74), and the excellent rate of collection was 71.6%(53/74). There was a significantly statistical differentce in the number of CD34+ cells in grafts collected from patients with chemotherapy cycle ≤6 and >6 ［(9.1±5.2)×106/kg vs (6.4±3.7)×106/kg, P=0.031］. The number of CD34+ cells in the first collection was positively correlated with WBC count, hemoglobin, platelet count, neutrophil count, lymphocyte count, monocyte count and hematocrit value on the day of collection ( r value was 0.424,0.486,0.306,0.289,0.353,0.428,0.528, respectively). WBC count, hemoglobin, monocyte count and hematocrit value have higher predictive value for the first collection of CD34+ cells. The area under the receiver operating characteristic was 0.7061,0.7845,0.7319,0.7848, respectively.@*CONCLUSION@#Low dose CTX and VP16 chemotherapy combined with G-CSF can effectively mobilize autologous peripheral blood stem cells. The cycle number of chemotherapy relates to the collection of autologous peripheral blood stem cells. After mobilization, the success of the first collection can be better predicted by the blood routine indexes.

Regulation of Neuro-Microenvironment on Mitochondrial Mass of Hematopoietic Stem and Progenitor Cells / 中国实验血液学杂志

OBJECTIVE@#To study the effects of the neuro-microenvironment on the mass of mitochondria in hematopoietic stem and progenitor cells (HSPC), and to understand the potential mechanisms how nerve regulates HSPC.@*METHODS@#6-hydroxydopamine (6-OHDA) and capsaicin were used to interfere with the function of sympathetic nerve and nociceptive nerve in mitochondria-GFP reporter mice, respectively. The fluorescence intensity of GFP in bone marrow and spleen was measured by flow cytometry. The GFP median fluorescence intensity (MFI) of HSPC in normal bone marrow and spleen was analyzed and compared. The changes of the mitochondrial mass in HSPCs in each group after denervation were compared.@*RESULTS@#Hematopoietic stem cells (HSC) had the highest mito-GFP MFI in steady-state (49 793±1 877), and the mito-GFP MFI gradually decreased during the differentiation of HSCs. Compared with control group, pharmaceutical nociceptive denervation significantly increased the mito-GFP MFI of bone marrow multipotent progenitor-1 (MPP1, 50 751±420 vs 44 020±510) and LKS- cells (15 673±65 vs 13 979±103); pharmaceutical sympathetic denervation significantly reduced the mito-GFP MFI of bone marrow LKS+ cells (21 667±351 vs 29 249±973).@*CONCLUSION@#Sympathetic and nociceptive nerves can regulate the mass of mitochondria in HSPC and affect the function of HSPCs.

Research Progress on the Mechanism of Macrophages Regulating Hematopoiesis in Bone Marrow Microenvironment--Review / 中国实验血液学杂志

Bone marrow macrophage is an important component of bone marrow microenvironment, which is closely related to hematopoietic regulation and hematopoietic stem cell transplantation(HSCT). Recent studies have shown that bone marrow macrophage is an important part of hematopoietic stem cell niche, which can help regulate the mobilization and function of hematopoietic stem/progenitor cells. After HSCT, the microenvironment of bone marrow is damaged and a large number of macrophages infiltrate into the bone marrow. Regulating the macrophage-related signal pathways can promote the recovery of hematopoiesis and the reconstruction of hematopoietic function. Co-culture of macrophages and hematopoietic stem cells (HSC) in vitro significantly increased the number of HSCs and their ability of clone formation, which suggests that macrophages play an important role in the regulation of hematopoiesis in the hematopoietic microenvironment of bone marrow. This paper reviews the recent research progress on the role of macrophages in bone marrow hematopoietic microenvironment.

Research Advances on Strategies to Promote Homing and Engraftment of Hematopoietic Stem Cells--Review / 中国实验血液学杂志

The homing and engraftment of hematopoietic stem cells (HSC) into bone marrow is the first critical step for successful clinical hematopoietic stem cell transplantation (HSCT). SDF-1 / CXCR4 is considered to be a very promising target to promote HSC homing. In recent years, with the in-depth research on the HSC homing, a variety of new strategies for promoting HSC homing and engraftment have been explored, such as nuclear hormone receptor, histone deacetylase inhibitor, prostaglandin and metabolic regulation, so as to increase the success rate of HSCT and improve the survival of patients. In this review, the recent research advances in the mechanism of HSC homing and strategies to promote HSC homing and engraftment were summarized and discussed.

Chronic Injury of Mice Bone Marrow Multipotent Hematopoietic Progenitor Cells Induced by Ionizing Radiation / 中国实验血液学杂志

OBJECTIVE@#To explore the chronic injury and its possible mechanism of ionizing radiation on multipotent hematopoietic progenitor cells (MPPs) by determining the related indicators of MPPs in bone marrow of mice post-radiation.@*METHODS@#Sixteen C57BL/6 adult mice were randomly divided into normal control and irradiation groups, 8 mice in each group. The mice in irradiation group were exposed to 6 Gy X-ray. The proportion of bone marrow MPPs, their apoptosis and proliferation 2 months after irradiation were detected by flow cytometry. Mitochondrial activity and levels of reactive oxygen species (ROS) in each MPPs population were detected by Mitotracker Red and DCFDA probes, and the senescent state of MPPs in the bone marrow was analyzed.@*RESULTS@#Ionizing radiation could reduce the proportion of MPPs in mouse bone marrow. The proportions and numbers of MPP1, MPP3 and MPP4 in the bone marrow were significantly decreased after whole-body irradiation with 6 Gy X-ray (P<0.05). In addition, radiation significantly reduced the colony-forming capacity of MPPs in bone marrow (P<0.05), the proportions of apoptotic cells in the MPP1 and MPP4 cell populations increased significantly in the bone marrow (P<0.05). The activity of mitochondria was significantly reduced in the bone marrow MPP2, MPP3 and MPP4 cell populations compared with that of the control group (P<0.05). It was also found that the radiation could significantly increase the ROS levels of MPPs in bone marrow, and the content of ROS in the MPP2, MPP3 and MPP4 cell population of the bone marrow was significantly increased(P<0.05). The senescent cells ratios of MPP1, MPP3 and MPP4 cells in the bone marrow after irradiation were significantly higher than those in the control group (P<0.05).@*CONCLUSION@#Ionizing radiation can cause chronic MPPs damage in mice, which is closely associated with persistent oxidative stress, cells apoptosis, and cellular senescence.

Effect of Recombinant Human Thrombopoietin (rhTPO) on Long-term Hematopoietic Recovery in Mice with Acute Radiation Sickness and Relative Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect and relative mechanism of Recombinant Human Thrombopoietin (rhTPO) on long-term hematopoietic recovery in mice with acute radiation sickness.@*METHODS@#Mice were intramuscularly injected with rhTPO (100 μg/kg) 2 hours after total body irradiation with 60Co γ-rays (6.5 Gy). Moreover, six months after irradiation, peripheral blood, hematopoietic stem cells (HSC) ratio, competitive transplantation survival rate and chimerization rate, senescence rate of c-kit+ HSC, and p16 and p38 mRNA expression of c-kit+ HSC were detected.@*RESULTS@#Six months after 6.5 Gy γ-ray irradiation, there were no differences in peripheral blood white blood cells, red blood cells, platelets, neutrophils and bone marrow nucleated cells in normal group, irradiated group and rhTPO group (P>0.05). The proportion of hematopoietic stem cells and multipotent progenitor cells in mice of irradiated group was significantly decreased after irradiation (P<0.05), but there was no significant changes in rhTPO group (P>0.05). The counts of CFU-MK and BFU-E in irradiated group were significantly lower than that in normal group, and rhTPO group was higher than that of the irradiated group(P<0.05). The 70 day survival rate of recipient mice in normal group and rhTPO group was 100%, and all mice died in irradiation group. The senescence positive rates of c-kit+ HSC in normal group, irradiation group and rhTPO group were 6.11%, 9.54% and 6.01%, respectively (P<0.01). Compared with the normal group, the p16 and p38 mRNA expression of c-kit+ HSC in the irradiated mice were significantly increased (P<0.01), and it was markedly decreased after rhTPO administration (P<0.01).@*CONCLUSION@#The hematopoietic function of mice is still decreased 6 months after 6.5 Gy γ-ray irradiation, suggesting that there may be long-term damage. High-dose administration of rhTPO in the treatment of acute radiation sickness can reduce the senescence of HSC through p38-p16 pathway and improve the long-term damage of hematopoietic function in mice with acute radiation sickness.

The effect of platelet infusion on the repair of bone marrow hematopoietic niche damage in mice induced by (60)Co radiation and hematopoietic reconstruction after bone marrow transplantation / 中华血液学杂志

Objective: To observe the effect of platelets on hematopoietic stem cell (HSCs) implantation in mice with radiation-induced bone marrow injury and bone marrow transplantation models. Methods: ①Male C57BL/6 mice were divided into a single irradiation group and a radiation infusion group after receiving (60)Co semimyeloablative irradiation for 18-10 weeks. The irradiation infusion group received 1×10(8) platelets expressing GFP fluorescent protein. ② The allogeneic bone marrow transplantation model was established. The experimental groups included the simple transplantation group (BMT) and the transplantation infusion group (BMT+PLT). The BMT group was infused through the tail vein only 5 × 10(6) bone marrow cells, the BMT+PLT group needs to be infused with bone marrow cells at the same time 1× 10(8) platelets. ③ Test indicators included peripheral blood cell and bone marrow cell counts, flow cytometry to detect the proportion of hematopoietic stem cell (HSC) and hematopoietic progenitor cells, bone marrow cell proliferation and apoptosis, and pathological observation of vascular niche damage and repair. Results: ①On the 3rd, 7th, 14(th), and 21st days after irradiation, the bone marrow cell count of the infusion group was higher than that in the single irradiation group (P<0.05), and the peripheral blood cell count was also higher. A statistically significant difference was found between the white blood cell count on the 21st day and the platelet count on the 7th day (P<0.05). In the observation cycle, the percentage of bone marrow cell proliferation in the infusion group was higher, while the percentage of apoptosis was lower. ② The results of bone tissue immunofluorescence after irradiation showed that the continuity of hematopoietic niche with red fluorescence was better in the irradiation infusion group. ③The chimerism percentage in the BMT+PLT group was always higher than that in the BMT group after transplantation.④ The BMT+PLT group had higher bone marrow cell count and percentage of bone marrow cell proliferation on the 7th and 28th day after transplantation than that in the BMT group, and the percentage of bone marrow cell apoptosis on the 14th day was lower than that in the BMT group (P<0.05). After the 14th day, the percentage of stem progenitor cells in the bone marrow cells of mice was higher than that in the BMT group (P<0.05). ⑤The immunohistochemical results of bone marrow tissue showed that the continuity of vascular endothelium in the BMT+PLT group was better than that in the BMT group. Conclusion: Platelet transfusion can alleviate the injury of vascular niche, promotes HSC homing, and is beneficial to hematopoietic reconstruction.

Expansion of cord blood stem cells in fibronectin-coated microfluidic bioreactor

ABSTRACT Background: Hematopoietic stem/progenitor cell transplantation is the main treatment option for hematological malignancies and disorders. One strategy to solve the problem of low stem cell doses used in transplantation is pre-transplant expansion. We hypothesized that using fibronectin-coated microfluidic channels would expand HSPCs and keep self-renewal potential in a three-dimensional environment, compared to the conventional method. We also compared stem cell homing factors expression in microfluidic to conventional cultures. Materials and methods: A microfluidic device was created and characterized by scanning electron microscopy. The CD133+ cells were collected from cord blood and purified. They were subsequently cultured in 24-well plates and microfluidic bioreactor systems using the StemSpan serum-free medium. Eventually, we analyzed cell surface expression levels of the CXCR4 molecule and CXCR4 mRNA expression in CD133+ cells cultured in different systems. Results: The expansion results showed significant improvement in CD133+ cell expansion in the microfluidic system than the conventional method. The median expression of the CXCR4 in the expanded cell was lower in the conventional system than in the microfluidic system. The CXCR4 gene expression up-regulated in the microfluidic system. Conclusion: Utilizing microfluidic systems to expand desired cells effectively is the next step in cell culture. Comparative gene expression profiling provides a glimpse of the effects of culture microenvironments on the genetic program of HSCs grown in different systems.

Aspectos citogenéticos y moleculares en los síndromes mielodisplásicos / Cytogenetic and molecular aspects in myelodysplastic syndromes

Introducción: Los síndromes mielodisplásicos constituyen un grupo heterogéneo de alteraciones de la célula progenitora hematopoyética. Estos se caracterizan por presentar una médula ósea hipercelular, una hematopoyesis inefectiva, displasia y citopenia periférica y la posibilidad de evolución a leucemia mieloide aguda. Objetivo: Describir las alteraciones citogenéticas y moleculares más frecuentes de los síndromes mielodisplásicos. Métodos: Se realizó una revisión de la literatura en los idiomas inglés y español, a través del sitio web PubMed y el motor de búsqueda Google académico, de artículos publicados en los últimos cinco años. Se realizó análisis y resumen de la bibliografía. Análisis y síntesis de la información: En los síndromes mielodisplásicos están presentes alteraciones citogenéticas frecuentes como la deleción de los cromosomas 5q, 7q y 20q, la monosomía del cromosoma 7, la trisomía del cromosoma 8 y la presencia de cariotipos complejos, que, unido a mutaciones somáticas en diferentes genes, intervienen en la patogénesis de la enfermedad y su conocimiento permite la estratificación pronóstica de los pacientes. Conclusiones: El diagnóstico a través de los estudios citogenéticos convencionales, la hibridación in situ por fluorescencia y la secuenciación génica permite una mayor comprensión de la biología de la enfermedad, la estratificación del riesgo y la toma de decisiones terapéuticas(AU)

Introduction: Myelodysplastic syndromes constitute a heterogeneous group of alterations of the hematopoietic progenitor cell, characterized by hypercellular bone marrow, ineffective hematopoietic, dysplasia and peripheral cytopenia; and the possibility of progressing to acute myeloid leukemia. Objective: To describe the most frequent cytogenetic and molecular alterations of myelodysplastic syndromes. Methods: A review of the literature in English and in Spanish was carried out, in the PubMed website and using the search engine Google, for articles published in the last five years. We performed analysis and summary of the reviewed bibliography. Analysis and synthesis of information: In myelodysplastic syndromes, frequent cytogenetic alterations are present such as deletion of chromosomes 5q, 7q and 20q, as well as the monosomy of chromosome 7, trisomy of chromosome 8 and the presence of complex karyotypes, which together with somatic mutations in different genes intervene in the pathogenesis of the disease and allow prognostic stratification of patients. Conclusions: Diagnosis through conventional cytogenetic studies, fluorescence in situ hybridization and gene sequencing allow a better understanding of the biology of the disease, risk stratification and therapeutic decision making(AU)

Validation of the single-platform ISHAGE protocol for enumeration of CD34+ hematopoietic stem cells in umbilical cord blood in a Brazilian center

Abstract Background This study aims to validate the single-platform method for enumeration of CD34+ cells, by comparing the performance of two different commercial kits, as well as to evaluate the efficiency of the AccuriTM C6 cytometer in providing direct counts of absolute cell numbers. Method We evaluated 20 samples from umbilical cord blood (UCB), comparing the two different methodologies for enumeration of CD34+ cells: single and dual-platform. For the assessment of the single-platform, Procount and SCE kits were used, both of which use fluorescent beads as a counting reference to obtain absolute CD34+ cells numbers. Moreover, after the acquisition of samples in flow cytometer AccuriTM C6, following the protocol established for each kit, the number of CD34+ cells was recalculated, considering the cell count provided by the AccuriTM C6. Main Results In our analysis, the results showed a strong correlation between the number of CD34+ cells/μL (r2 = 0.77) when comparing the SCE kit and the current dual-platform method. On the other hand, the comparison between Procount kit and dual-platform results showed a moderate correlation for the number of CD34+/μL cells (r2 = 0.64). Conclusion Our results showed that the AccuriTM C6 flow cytometer can be used safely, applying both the dual and single platform analysis strategy. Considering the ISHAGE protocol-based single-platform approach, as the most appropriate methodology for CD34+ cells enumeration, our results demonstrated that the SCE kit has great potential for national standardization of UCB samples analysis methodology.

SARS-COV-2 screening in allogeneic hematopoietic stem cell donors: Implications for the evaluation process and eligibility

Abstract Introduction Soon after the onset of the SARS-CoV-2 pandemic, viral screening by nasopharyngeal swab became mandatory for allogeneic hematopoietic stem cell (HSC) donor eligibility. Methods We described our monocenter experience with allogeneic HSC donors from February 1 to the October 31, 2020 to verify whether the introduction of SARS-CoV-2 screening altered the donor eligibility and/or entailed a prolongation of the evaluation process. Results A total of 21 allogeneic HSC donors were screened during the above-mentioned period upon request by the local transplant physicians or by the Italian Bone Marrow Donor Registry; among the HSC donors (n = 17) who completed the eligibility process and further received the nasopharyngeal swab, all but one were negative for the presence of SARS-CoV-2. The positive donor remained asymptomatic for the whole duration of the infection, which lasted six weeks. However, he was temporarily excluded from donation. The median duration of the evaluation process was not significantly different, compared to the same period of 2019 (p-value = 0.11). Conclusion The mandatory SARS-CoV-2 screening in allogeneic HSC donors allowed for the detection of 6% positivity in this monocenter series over a 9-month period. Despite the inconvenience of this unexpected non-eligibility, the exclusion of a SARS-CoV-2 positive donor represented an important safety measure for the donor, with respect to a new and still partially unknown virus. The screening did not alter the length of the donor evaluation and thus, did not cause a delay in the eligibility process.

Análise dos fatores associados ao retransplante de células-tronco hematopoiéticas: estudo caso-controle / Analysis of factors associated with hematopoietic stem-cell retransplantation: a case-control study / Análisis de factores asociados al Retrasplante de Células Madre Hematopoyéticas: un estudio de caso-control

Resumo Objetivo: analisar os fatores associados ao insucesso do Transplante de Células-Tronco Hematopoiéticas (TCTH) em pacientes submetidos ao retransplante de Células-Tronco Hematopoiéticas (RCTH). Método: estudo quantitativo do tipo caso-controle para avaliar pacientes submetidos ao RCTH. Para tanto, utilizou-se amostra pareada de dois controles para cada caso (2:1). O grupo caso foi constituído pelos prontuários de saúde com todos os pacientes que foram submetidos ao RCTH (28) e o grupo controle (56) incluiu pacientes que receberam apenas um transplante. Três variáveis nortearam o pareamento: sexo, diagnóstico e tipo de transplante. Resultados: vinte e quatro (85,71%) pacientes do grupo caso receberam retransplante devido a recidiva da doença e quatro (14.29%) devido a falha do enxerto. Uma diferença estatística foi encontrada na análise entre os pacientes que não usaram o ácido ursodesoxicólico, analgésicos opioides ou imunossupressores. A necessidade de um RCTH entre aqueles que usaram estes medicamentos de forma inapropriada foi 16,12, 12,79 e 4,5 vezes maior, respectivamente, do que entre os que as usaram corretamente. Conclusão: houve uma diferença relacionada ao motivo que levou ao retransplante e os indivíduos analisados. A conclusão é que a razão preditiva para retransplante nesta amostra foi a recidiva da doença.

Abstract Objective: to analyze the factors associated with the failure of Hematopoietic Stem Cell Transplantation (HSCT) in patients undergoing Hematopoietic Stem Cell Retransplantation (HSCR). Method: this study implemented a quantitative approach and was a case-control type which addressed patients undergoing HSCR. To do so, a paired sample of two controls was used for each case (2:1). The case group consisted of the medical records of all patients who underwent HSCR (28) and the control group (56) of those who underwent only one transplant. Three variables guided the pairing: gender, diagnosis and type of transplant. Results: a total of 24 (85.71%) patients in the case group were re-transplanted due to disease relapse and four (14.29%) due to graft failure. There was a statistical difference in the analysis between patients who did not use ursodeoxycholic acid, opioid analgesics and immunosuppressants. The need for HSCR among those who used these medications inappropriately was 16.12, 12.79 and 4.5 times more likely, respectively, than those who used them correctly. Conclusion: there was a difference regarding the reasons which led to the retransplantation and the analyzed subjects, and this study concluded that the predictive reason for retransplantation in the studied sample was disease relapse.

Resumen Objetivo: analizar los factores asociados con el fracaso del Trasplante de Células Madre Hematopoyéticas (TCMH) en pacientes sometidos al Retrasplante de Células Madre Hematopoyéticas (RCMH). Método: estudio cuantitativo de tipo caso-control que abordó pacientes sometidos al RCMH. Para esto, se utilizó una muestra pareada de dos controles para cada caso (2:1). El grupo caso estuvo formado por los registros médicos de todos los pacientes que fueron sometidos al RCMH (28) y el grupo control (56) por los que fueron sometidos a un solo trasplante. Tres variables guiaron el emparejamiento: género, diagnóstico y tipo de trasplante. Resultados: un total de 24 (85.71%) pacientes en el grupo caso fueron retransplantados debido a la recaída de la enfermedad y 4 (14.29%) por el fracaso del injerto. Hubo una diferencia estadística en el análisis entre los pacientes que no usaron ácido ursodesoxicólico, analgésicos opioides e inmunosupresores. La necesidad de RCMH entre los que usaron estos medicamentos de manera inapropiada se encontraba 16,12 - 12,79 y 4,5 veces más probable, respectivamente, que aquellos que los usaron correctamente. Conclusión: hubo diferencia en cuanto a las razones que llevaron al retrasplante de los sujetos analizados. Este estudio concluyó que la razón predictiva del retrasplante, en la muestra estudiada, fue la recidiva de la enfermedad.

Role of DNMT3a in Hydroquinone-Induced Hematopoietic Stem Cell Toxicity / 中国实验血液学杂志

OBJECTIVE@#To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity.@*METHODS@#Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively.@*RESULTS@#Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001).@*CONCLUSION@#DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.

A Single-Center Analysis of the Use of G-CSF Combined with Plerixafor to Mobilize Peripheral Blood Hematopoietic Stem Cell from Healthy Related Donors in Allogeneic Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

OBJECTIVE@#To study the effect and safety of G-CSF combined with Plerixafor on the mobilization of peripheral blood hematopoietic stem cells from healthy related donors of allogeneic hematopoietic stem cell transplantation (allo-HSCT).@*METHODS@#It was analyzed retrospectively that the data of peripheral blood hematopoietic stem cells from 33 (observation group) related donors mobilized by G-CSF plus Plerixafor in Hebei Yanda Lu Daopei Hospital from April 2019 to April 2021. Bone marrow and peripheral blood hematopoietic stem cells (PBSCs) of these donors were respectively collected on the fourth and fifth day of G-CSF-induced mobilization. Following the administration of Plerixafor on the night of the fifth day, PBSCs were collected on the sixth day once again. 46 donors using "G-CSF only" mobilization method in the same period were randomly selected as the control and respectively analyzed the differences of CD34+ cell counts on the fifth and the sixth day in two groups. And the donors' adverse reaction to Plerixafor in the form of questionnaire was also observed. Then it was compared that the patients who underwent allo-HSCT in "G-CSF+Plerixafor" group and "G-CSF only" group in terms of acute GVHD at grade I-IV or III-IV, CMV reactivation and EBV reactivation.@*RESULTS@#CD34+ cells count (M±Q) among PBSCs collected on the fifth and the sixth day in the observation group were (1.71±1.02)×106/kg and (4.23±2.33)×106/kg, respectively. CD34+ cell counts on the sixth day was significantly higher than that of the fifth day (P<0.001); While the counterparts in the control group were (2.47±1.60)×106/kg and (1.87±1.37)×106/kg, respectively. By statistical analysis, CD34+ cell counts on the sixth day was significantly less than that of the fifth day (P<0.001). The adverse reaction to Plerixafor for the donors in the study were all grade 1 or 2 (mild or moderate) according to CTCAE 5.0 and disappeared in a short time. The patients who underwent allo-HSCT in the "G-CSF+Plerixafor" group and "G-CSF only" group were not statistically significant in terms of acute GVHD at grade I-IV or III-IV, CMV reactivation and EBV reactivation (P>0.1).@*CONCLUSION@#The cell mobilization program of G-CSF combined with Plerixafor is safe and effective for being applied to allo-HSCT. The addition of Plerixafor can significantly increase the number of CD34 postive cells in the PBSC collection. Key words　　; ;

Hematopoyesis Clonal de Potencial Indeterminado (HCPI): más allá de un modelo de campo de cancerización / Clonal hematopoiesis of indeterminate potential (CHIP): Beyond a model of field cancerization

Resumen La Hematopoyesis Clonal de Potencial Indeterminado (HCPI), más conocida como CHIP por sus siglas en inglés, se define como la expansión clonal de Células Madre Hematopoyéticas (CMHs) que albergan una o más mutaciones somáticas (en la mayoría de los casos una sola mutación) sin un cáncer hematológico subyacente ni evidencia morfológica definitiva de displasia, con una frecuencia alélica mayor al 2%. Los individuos con HCPI progresan a malignidad a una tasa de cerca del 0.5% a 1% por año, convirtiéndose así en un modelo de campo de cancerización. Sin embargo, sus implicaciones van más allá debido a que se ha encontrado asociación con enfermedades inflamatorias crónicas, como enfermedad cardiovascular ateroesclerótica, diabetes y enfermedades autoinmunes. Además, es considerado un factor predictivo en pacientes con cáncer hematolológico y no hematológico que reciben quimioterapia y radioterapia.

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is the expansion of hematopoietic stem cells harboring one or more somatic mutations. These patients do not have underlying hematologic neoplasia, myelodysplasia, or dysplasia, but can progress to a malignant state at a rate of 0.5 to 1% per year. CHIP could be used as a model of field cancerization, since it has been associated with chronic inflammatory diseases, arteriosclerosis, diabetes, and autoimmune conditions. CHIP is also considered a predictive factor in hematological and non-hematological cancer patients receiving chemotherapy and radiotherapy.

Generation of hematopoietic stem/progenitor cells with sickle cell mutation from induced pluripotent stem cell in serum-free system

ABSTRACT Introduction Sickle cell disease (SCD) is a monogenic disease and it is estimated that 300,000 infants are born annually with it. Most treatments available are only palliative, whereas the allogeneic hematopoietic stem cell transplantation offers the only potential cure for SCD. Objective Generation of human autologous cells, when coupled with induced pluripotent stem cell (iPSC) technology, is a promising approach for developing study models. In this study, we provide a simple and efficient model for generating hematopoietic cells using iPSCs derived from a sickle cell anemia patient and an inexpensive in-house-prepared medium. Method This study used iPSCs previously generated from peripheral blood mononuclear cells (PBMCs) from a patient with sickle cell anemia (iPSC_scd). Hematopoietic and erythroid differentiation was performed in two steps. Firstly, with the induction of hematopoietic differentiation through embryoid body formation, we evaluated the efficiency of two serum-free media; and secondly, the induction of hematopoietic stem/progenitor cells to erythroid progenitor cells was performed. Results The patient-specific cell line generated CD34+/CD45+ and CD45+/CD43+ hematopoietic stem/progenitor cells and erythroid progenitors, comprising CD36+, CD71+ and CD235a+ populations, as well as the formation of hematopoietic colonies, including erythroid colonies, in culture in a semi-solid medium. Conclusion In conjunction, our results described a simple serum-free platform to differentiate human the iPSCs into hematopoietic progenitor cells. This platform is an emerging application of iPSCs in vitro disease modeling, which can significantly improve the search for new pharmacological drugs for sickle cell disease.

Optimización de unidades formadoras de colonias y detección del virus de papiloma humano en sangre de cordón umbilical / Colony-forming units optimization and human papillomavirus detection in umbilical cord blood

Resumen Introducción: Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos. Objetivo: Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU. Métodos: Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming. Resultados: La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p < 0.0001); así como la de SCU comparada con la de segmento (p < 0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+. Conclusiones: Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

Abstract Introduction: Analysis of several markers is required for adequate quality control in umbilical cord blood units (UCBU) when are used for therapeutic purposes. Objective: To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU. Methods: One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the SSP technique. Results: CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p < 0.0001), as well as that of UCB in comparison with that of the segment (p < 0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY11 and GP5/GP6+. Conclusions: Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.