Measuring ATP Concentration in a Small Number of Murine Hematopoietic Stem Cells

Author(s):  
Krzysztof Szade ◽  
Monika Zukowska ◽  
Alicja Jozkowicz ◽  
Jozef Dulak
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hongmei Li ◽  
Bowen Hu ◽  
Shang Hu ◽  
Wen Luo ◽  
Donglei Sun ◽  
...  

Abstract Background B-cell CLL/lymphoma 6 (BCL6) is a transcriptional master regulator that represses more than 1200 potential target genes. Our previous study showed that a decline in blood production in runting and stunting syndrome (RSS) affected sex-linked dwarf (SLD) chickens compared to SLD chickens. However, the association between BCL6 gene and hematopoietic function remains unknown in chickens. Methods In this study, we used RSS affected SLD (RSS-SLD) chickens, SLD chickens and normal chickens as research object and overexpression of BCL6 in hematopoietic stem cells (HSCs), to investigate the effect of the BCL6 on differentiation and development of HSCs. Results The results showed that comparison of RSS-SLD chickens with SLD chickens, the BCL6 was highly expressed in RSS-SLD chickens bone marrow. The bone marrow of RSS-SLD chickens was exhausted and red bone marrow was largely replaced by yellow bone marrow, bone density was reduced, and the levels of immature erythrocytes in peripheral blood were increased. At the same time, the hematopoietic function of HSCs decreased in RSS-SLD chickens, which was manifested by a decrease in the hematopoietic growth factors (HGFs) EPO, SCF, TPO, and IL-3, as well as hemoglobin α1 and hemoglobin β expression. Moreover, mitochondrial function in the HSCs of RSS-SLD chickens was damaged, including an increase in ROS production, decrease in ATP concentration, and decrease in mitochondrial membrane potential (ΔΨm). The same results were also observed in SLD chickens compared with normal chickens; however, the symptoms were more serious in RSS-SLD chickens. Additionally, after overexpression of the BCL6 in primary HSCs, the secretion of HGFs (EPO, SCF, TPO and IL-3) was inhibited and the expression of hemoglobin α1 and hemoglobin β was decreased. However, cell proliferation was accelerated, apoptosis was inhibited, and the HSCs entered a cancerous state. The function of mitochondria was also abnormal, ROS production was decreased, and ATP concentration and ΔΨm were increased, which was related to the inhibition of apoptosis of stem cells. Conclusions Taken together, we conclude that the high expression of BCL6 inhibits the differentiation and development of HSCs by affecting mitochondrial function, resulting in impaired growth and development of chickens. Moreover, the abnormal expression of BCL6 might be a cause of the clinical manifestations of chicken comb, pale skin, stunted growth and development, and the tendency to appear RSS in SLD chickens.


2006 ◽  
Author(s):  
Hideyo Hirai ◽  
Pu Zhang ◽  
Tajhal Dayaram ◽  
Christopher Hetherington ◽  
Shin-ichi Mizuno ◽  
...  

Author(s):  
И.Ю. Маклакова ◽  
Д.Ю. Гребнев ◽  
А.П. Ястребов

Цель - изучение влияния сочетанной трансплантации мультипотентных мезенхимальных стромальных (ММСК) и гемопоэтических стволовых клеток (ГСК), выделенных из плаценты, на регенерацию белой и красной пульпы селезенки в физиологических условиях и в условиях воздействия ионизующего излучения. Методика. Эксперименты выполнены белых лабораторных беспородных мышах-самцах. Облучение животных проводилось на гамма-терапевтической установке типа АГАТ-С с радионуклидным источником Co-60 типа ГИК-8-4, поглощенная доза составила 4,0 Гр, мощность поглощенной дозы 20 сГр/мин. Животным опытной группы внутривенно вводились аллогенные ММСК и ГСК соответственно в дозе 6 млн клеток/кг и 330 тыс. клеток/кг, суспендированные в 0,2 мл 0,9% раствора NaCl. Выделение гемопоэтических стволовых клеток осуществлялось методом прямой иммуномагнитной сепарации. Проводили морфометрию лимфоидных фолликулов селезенки (средняя площадь, средняя площадь В-зоны, средняя площадь герминативного центра, средняя площадь T-зоны), а также определялось среднее расстояние между центрами фолликулов и средняя клеточность красной пульпы. Результаты. Показано, что после воздействия ионизирующего излучения на фоне сочетанной трансплантации ММСК и ГСК происходит увеличение размеров лимфоидного фолликула за счет площади B-зоны фолликула, площади герминативного центра фолликула, восстановление содержания лимфобластов, пролимфоцитов и лимфоцитов до значений нормы. На фоне трансплантации ММСК и ГСК в условиях лучевой нагрузки установлено увеличение плотности клеток в красной пульпе селезенки и, как следствие, увеличение расстояния между центрами лимфоидных фолликулов. Увеличение плотности клеток в красной пульпе происходит как за счет увеличения содержания эритроидных клеток, так и за счет увеличения гранулоцитов. Заключение. Проведенные исследования свидетельствуют об эффективности сочетанной трансплантации ММСК и ГСК в отношении основных морфометрических показателей селезенки после воздействия ионизирующего излучения. The purpose of this work was to study the effect of combined transplantation of multipotent mesenchymal stromal (MSCS) and hematopoietic stem cells (HSCs) isolated from the placenta, on the regeneration of white and red pulp of the spleen under physiological conditions and in conditions of exposure to ionizing radiation. Methods. The experiments were performed with laboratory mice-males. We studied the influence of ionizing radiation dose of 4.0 Gy. Animals of the experimental group were intravenously infused into MMSC and GSK respectively at a dose of 6 million cells/kg and 330 thousand cells/kg, suspended in 0.2 ml of 0.9% NaCl solution. The selection of hematopoietic stem cells was carried out using the direct technique of immune magnetic separation. Were studied the following morphometric parameters of the spleen: the average area of lymphoid follicles, the average area of zone of lymphoid follicles, average size of germinal center of lymphoid follicles, average size T-zones of lymphoid follicles, the average distance between the centers of the follicles, the average cellularity of the red pulp. Results. As a result, of research obtained that after exposure to ionizing radiation on the background of combined transplantation of HSC and MSCS there is an increase in size of lymphoid follicle at the expense of area B-zone of the follicle, the area germinative center of the follicle, restoring the content of lymphoblasts and lymphoblasts and lymphocytes to normal values. On the background of transplantation MMSC and GSK in terms of radiation exposure changes and the red pulp of the spleen. The increase in the density of cells in the red pulp of the spleen and, as a consequence, of the increase of the distance between the centers of lymphoid follicles. The increase in the density of cells in the red pulp occurs due to the increase in the content of erythroid cells and by increasing granulocytes. Key words: ionizing radiation, multipotent mesenchymal stromal cells, hematopoietic stem cells, spleen, regeneration. Conclusion. Studies have shown the effectiveness of combined transplantation MSC and GSK in respect of the main morphometric parameters of the spleen after exposure to ionizing radiation.


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