CN113567407B

CN113567407B - Method for detecting mitochondrial function of hematopoietic cells

Info

Publication number: CN113567407B
Application number: CN202110845352.7A
Authority: CN
Inventors: 梁昊岳; 付伟超; 于文颖; 高瀛岱
Original assignee: Institute of Hematology and Blood Diseases Hospital of CAMS and PUMC
Current assignee: Institute of Hematology and Blood Diseases Hospital of CAMS and PUMC
Priority date: 2021-07-26
Filing date: 2021-07-26
Publication date: 2024-02-27
Anticipated expiration: 2041-07-26
Also published as: CN113567407A

Abstract

The invention relates to a method for detecting mitochondrial function of hematopoietic cells, which comprises the following steps: the mitochondrial quality, mitochondrial membrane potential, mitochondrial reactive oxygen species and mitochondrial autophagy levels of normal hematopoietic cells and/or pathological hematopoietic cells, including hematopoietic stem cells, hematopoietic progenitor cells and mature blood cells, are simultaneously detected and simultaneously assessed. Provides a complete set of methods and basic reference values for systematically studying mitochondrial metabolic functions.

Description

Method for detecting mitochondrial function of hematopoietic cells

Technical Field

The invention relates to the technical field of biology, in particular to a method for detecting and evaluating mitochondrial functions of hematopoietic cells.

Background

Hematopoietic cell lineage differentiation is an indispensable step in the development and formation of the hematopoietic system, and hematopoietic cells thus produced are involved in vital activities such as nutrient transport, immunity, hemostasis and wound healing of living bodies. Researchers have demonstrated by transplantation and colony analysis that hematopoietic stem cells (Hematopoietic stem cell, HSCs) are the origin of all blood cells and immune cells. The mammalian hematopoietic system begins with multipotent hematopoietic stem cells that differentiate first into multipotent progenitor cells (Pluripotent progenitor cells, MPPs), which do not have self-renewal capacity. MPPs differentiate rapidly into hematopoietic progenitor cells (Hemopoietic progenitor cells, HPCs) which have a strong multipotent differentiation capacity. Subsequently, the first bifurcation of the differentiation process occurs in common myeloid progenitor cells (Common myeloid progenitor cells, CMPs) and common lymphoid progenitor cells (Common lymphatic progenitor cells, CLPs), where CMPs have myeloid, erythroid and megakaryocyte differentiation potential, and CLPs have only lymphocyte differentiation potential. The second branch point of the differentiation process separates granulocyte-macrophage progenitor cells (Granulocyte macrophage progenitor cells, GMPs) and Megakaryocyte-erythroid progenitor cells (MEPs) at CMPs, which differentiate directly further into more mature B cells, T cells and NK cells. Whereas GMPs are subdivided into mature granulocytes and monocytes, MEPs produce megakaryocytes and erythrocytes.

Mitochondria are one of the most important organelles within cells, involved in the production of ATP, maintaining calcium balance, regulating reactive oxygen species production, and in signaling, inflammation, and cell death, among others. Alterations in mitochondrial function, such as oxidative phosphorylation injury, abnormal energy metabolism, inhibition of apoptosis, autophagy disorders, promotion of immune evasion and altered signaling pathways, may affect the occurrence of disease. The occurrence and development of human diseases such as hematological diseases, neuromuscular diseases, cardiovascular diseases, diabetes, tumors, etc. are closely related to mitochondrial activity. Cells at different stages of hematopoietic lineage differentiation have differences in mitochondrial function, and mitochondrial mass (Mitochondrial mass, MM), mitochondrial membrane potential (Mitochondrial membrane potential, MMP), mitochondrial reactive oxygen species (Reactive oxygen species, ROS), and mitochondrial autophagy levels can be used as main indicators to reflect the role of mitochondria in a series of physiological and pathological processes involved in cell activity, stress, aging, and the like.

Researchers generally believe that MM is increased during hematopoietic stem cell differentiation and lineage development. For HSCs, mitochondrial biogenesis and ROS levels are inhibited by the TSC 1-mediated mTOR pathway, thus maintaining the resting state and function of HSCs, a process that is accompanied by a decrease in MM and a decrease in ROS. In the mouse model, LSK (Lin ^- Sca1 ⁺ c-Kit ⁺ ) Increased MM in cells results in long-term regeneration. MM in mouse HSCs was found to be similar to, but lower than, the metamedullary differentiated progenitor cells. At the same time, researchers have found that tumor cells exhibit high MMPs relative to healthy cells, and that cells with higher MMPs continue to divide and form tumors more easily, while lower MMP cells differentiate more efficiently into other cell types by transplanting subpopulations of embryonic stem cells into mice. ROS have damaging effects on DNA, lipids, and proteins, indicating that mitochondrial respiratory chain dysfunction and inefficient oxidative phosphorylation (Oxidative phosphorylation, OXPHOS) may lead to more electron leakage andfurther increases in ROS production result in deleterious circulation, ultimately causing irreversible damage to the cell. It was found that ROS appear to play a positive and necessary signaling role in stem cell biology, with the production of small amounts of ROS being important signaling molecules for cell activation, which act as signal messengers in a wide variety of cellular processes. At the same time, decreasing mitochondrial activity by decoupling the electron transfer chain (Electron transport chain, ETC) stimulates autophagy and drives self-renewal of HSCs, preserving their ability to differentiate properly and functional blood remodelling. In addition, autophagy maintains HSC function in aged mice and is likely to be involved in metabolic switching during iPSC reprogramming.

The mitochondrial function detection method of the hematopoietic cells with more application at present mainly comprises mitochondrial quality detection, mitochondrial membrane potential detection, mitochondrial active oxygen detection, mitochondrial autophagy detection and the like. Wherein the detection of mitochondrial mass can be performed by fluorescence probe MitoTracker Green FM in combination with flow cytometry. MitoTracker Green FM contains a chloromethyl functional group labeled with weak thiol reactivity of mitochondria, which hardly fluoresces in aqueous solution, and only fluoresces when accumulated in the lipid environment of mitochondria, and background fluorescence is almost negligible. MitoTracker Green FM's ability to localize to mitochondria is not affected by mitochondrial membrane potential, making it possible as a tool for quantifying mitochondrial mass. The detection of mitochondrial membrane potential mainly adopts a method of combining fluorescent dye probe methods such as rhodamine 123 (rhodi 123), tetramethyl rhodamine methyl ester (tetramethylrhodamine methyl ester, TMRM), tetramethyl rhodamine ethyl ester (tetramethylrhodamine ethyl ester, TMRE), tetrachloro tetraethyl benzimidazole carbonyl cyanine iodide (JC-1) and MitoTracker Red CMXRos with flow cytometry. The level of mitochondrial active oxygen can be measured using the fluorescent probe DCHF-DA dye; DCHF-DA itself is not fluorescent and can freely pass through cell membranes, and after entering cells, intracellular esterases hydrolyze to generate DCHF, which cannot enter and exit the cell membranes, so that probes can be easily marked in the cells. In the presence of active oxygen, DCHF is oxidized to form fluorescent material DCF, the fluorescence intensity of which is proportional to the intracellular active oxygen level, and the level of the active oxygen can be known by detecting the fluorescence of DCF. The mitochondrial active oxygen can be detected by using the fluorescent probe Mitosox, and the detection method has the advantages of simplicity, convenience, sensitivity, low background, wide linear range, short time and high detection efficiency. Mitochondrial autophagy can be detected by combining the fluorescent Dye Mitophagy Dye with flow cytometry, and Mtphagy Dye is combined on mitochondria in cells through chemical bonds, so that weaker fluorescence is maintained. By inducing mitochondrial autophagy, the mitochondrial autophagy will fuse with lysosomes and the fluorescence intensity of Mtphagy Dye will increase.

The flow cytometry is mainly applied to detection and analysis of cell phenotype, cell cycle, apoptosis, cell proliferation and the like, and has very wide application in hematology and stem cell research. However, the current studies of mitochondrial function of hematopoietic cells have limitations, mainly focusing on mitochondrial function in single or few types of hematopoietic cells, and lack of complete detection and simultaneous comparison of mitochondrial metabolic function of different types of normal and pathological hematopoietic cells under the same experimental conditions and within the same system standard. When flow cytometry is applied to detect mitochondrial function, complete experimental protocols including antibody collocation, instrument setup, detection time selection, photomultiplier voltage setting, and fluorescence compensation adjustment have not been developed. Due to the lack of simultaneous comparative studies of mitochondrial function of hematopoietic cells at different differentiation stages of the system, the failure to develop methods for simultaneous detection and simultaneous evaluation of mitochondrial function of hematopoietic stem cells, hematopoietic progenitor cells and mature blood cells based on flow cytometry has restricted the progress of research into the development of hematopoietic systems and differentiation of hematopoietic cells in model organisms.

Disclosure of Invention

The invention aims to overcome the defects of the prior art in application, and establishes an evaluation method of the mitochondrial function of hematopoietic cells based on flow cytometry, thereby forming a complete experimental scheme including antibody collocation, instrument setting, detection time selection, photomultiplier voltage setting and fluorescence compensation adjustment.

The invention adopts the technical scheme that:

the invention provides a method for detecting mitochondrial function of hematopoietic cells, which comprises the following steps: the mitochondrial quality, mitochondrial membrane potential, mitochondrial reactive oxygen species and mitochondrial autophagy levels of normal hematopoietic cells and/or pathological hematopoietic cells, including hematopoietic stem cells, hematopoietic progenitor cells and mature blood cells, are simultaneously detected and simultaneously assessed.

The normal hematopoietic cells include: HSC, MPP, HPC, CMP, GMP, MEP, CLP, monocytes, granulocytes, erythrocytes, B cells, T cells and NK cells. (wherein HSC, MPP and HPC are collectively referred to as HSPC, CMP, GMP and MEP are collectively referred to as committed HPC, B cells, T cells and NK cells are collectively referred to as lymphocytes.)

The pathological hematopoietic cells include: leukemia Stem Cells (LSC) and leukemia cells (non-LSC), in contrast to pathological cells, are normal primitive hematopoietic cells (LSK) and normal mature hematopoietic cells (non-LSK).

The simultaneous detection includes: the same flow cytometry is adopted, the same time point is used for detection, and the incubation time of fluorescent dye is the same, namely, the mitochondrial mass Mitotracker Green, mitochondrial membrane potential Mitothecker Red, mitochondrial active oxygen Mitosox Red and mitochondrial autophagy MitophagyDye are respectively incubated for 20-30min at 37 ℃ in a dark place; the photomultiplier voltages of the mitochondrial function detection fluorescent dye are respectively consistent in each flow template.

The simultaneous evaluation includes: using FlowJo ^TM v10.6.1 (BD Biosciences, usa) software acquires the flow data and subtracts the spectral overlap interference between the individual fluorescent channels from each other according to the multiparameter fluorescence compensation adjustment principle.

The invention has the beneficial effects that:

the method compares the mitochondrial functions of normal hematopoietic cells and pathological hematopoietic cells, and forms the simultaneous detection and simultaneous evaluation of the mitochondrial functions of the normal hematopoietic cells and the pathological hematopoietic cells including hematopoietic stem cells, hematopoietic progenitor cells and mature blood cells by optimizing mitochondrial quality, mitochondrial membrane potential, mitochondrial active oxygen and mitochondrial autophagy experimental schemes, thereby providing a complete set of methods and basic reference values for systematically researching mitochondrial metabolism functions.

The method can also combine single cell sequencing technology at the same time, performs preliminary analysis on the heterogeneity of the mitochondrial autophagy pathway of the hematopoietic stem progenitor cells, and lays a foundation for researching the role of mitochondrial function in blood diseases, cardiovascular diseases, tumors and aging in future.

Description of the drawings:

FIG. 1 is a diagram of hematopoietic cell differentiation grade;

FIG. 2 is a flow chart of a mitochondrial function study of hematopoietic cells;

FIG. 3 shows the ultrastructural features of hematopoietic cells at different differentiation stages, a.HSC, HPC and lineage committed HPCs b.Material myeloidcells c.Material lysis cells.

FIG. 4 is a graph showing the clustering results of mouse hematopoietic cells tSNE, flowSOM; wherein a.HSPC tSNE analysis b.HSPC flowSOM heat map c.HSPC flowSOM cluster analysis d.packaged HPC tSNE analysis e.packaged HPC flowSOM heat map f.packaged HPC flowSOM cluster analysis g.granulocyte and monocyte tSNE analysis h.granulocyte and monocyte flowSOM heat map i.granulocyte and monocyte flowSOM cluster analysis j.erythrocyte tSNE analysis k.erythrocyte flowSOM heat map i.erythrocyte flowSOM cluster analysis m.lymphocyte tSNE analysis n.lymphocyte flowSOM cluster analysis.

FIG. 5 is a graph of MM and MMP levels of hematopoietic cells at various stages of differentiation; wherein MM characteristic of a hematopoietic cell a, myeloid hematopoietic cell B, gonococcal hematopoietic cell C, hematopoietic progenitor cell d, mature hematopoietic cell B, SSC number of hematopoietic cell population a, myeloid hematopoietic cell B, gonococcal hematopoietic cell C, hematopoietic progenitor cell d, MMP level of mature hematopoietic cell C, myeloid hematopoietic cell B, gonococcal hematopoietic cell C, hematopoietic progenitor cell d, mature hematopoietic cell.

FIG. 6 is a graph of ROS and mitophagy levels of hematopoietic cells at various stages of differentiation, wherein ROS levels of A hematopoietic cells a. Myeloid hematopoietic cells B. Gonococcal cells c. Mature hematopoietic cells B hematopoietic cells mitophagy characteristics a. Myeloid hematopoietic cells B. Gonococcal cells c. Hematopoietic progenitor cells d. Mature hematopoietic cells.

FIG. 7 is a diagram of single cell sequencing analysis of mouse bone marrow hematopoietic stem/progenitor cells; HSC tSNE clustering results b.HSC autophagy related gene expression profile c.HSC autophagy related gene expression heat map d.committed HPC tSNE clustering results e.committed HPC autophagy related gene expression profile f.committed HPC autophagy related gene expression heat map.

FIG. 8 shows hematopoietic cell heterogeneity at the same stage of differentiation; wherein the ultrastructural view of a.hpc b.mep c.gmp d.erythyocyte e.cd4+t f.B cells B hematopoietic cell populations rSD a.myeloid hematopoietic cells b.gonococcal hematopoietic cells c.hematopoietic progenitor cells d.mature hematopoietic cells.

Fig. 9 is a graph of mitochondrial function levels of pathological cells versus normal hematopoietic cells, where a.ros b.mmp c.mitophagy.

FIG. 10 is a schematic diagram of 16 mouse hematopoietic cell flow-sorting a. Hematopoietic Stem Cells (HSCs), hematopoietic progenitor cells (HPC 1 and HPC 2) and pluripotent progenitor cells (MPPs); b. committed hematopoietic progenitors (CMP, GMP, and MEP); c. erythrocytes (EryA and EryB); d. granulocytes; e. monocytes; f. gonomic progenitor Cells (CLP); g.B cells, CD4+ T cells, CD8+ T cells and NK cells.

Detailed Description

Mitochondria are main places for biological oxidation and capacity conversion of cells, organisms take in oxygen through a respiratory system and a circulatory system and transfer the oxygen to the cells, ATP is finally generated in online granules through fatty acid, tricarboxylic acid circulation, ketone generation, oxidative phosphorylation and the like so as to supply energy required by life activities of the organisms, and more than nine times of energy consumption of the organisms are from the oxidative phosphorylation of the mitochondria, so that the mitochondria are known as a power plant of the cells. In addition, mitochondria are also the platform of intracellular signaling, regulators of innate immunity, and regulators of stem cell activity. Mitochondria play an important role in energy metabolism, signal transduction, cell aging and the like by generating ATP, maintaining the balance of calcium, regulating the generation of active oxygen, participating in signal transmission, inflammation, cell death and the like. Mitochondria can also coordinate immunity by regulating the metabolic and physiological states of different types of immune cells, where mitochondria can also regulate cell development, activation, proliferation, differentiation, and death.

In the basic and clinical research of blood system diseases, promoting the apoptosis of leukemia cells is one of the main measures for leukemia treatment, and the apoptosis 'mitochondrial pathway' opens up the field of leukemia treatment by taking mitochondria as a target spot. Adenine deoxynucleotide analogues and the like can change the permeability of mitochondrial membranes, release the related active substances of the apoptosis in mitochondria and start the Caspase cascade reaction of the apoptosis. The oxidative damage of tumor cells is increased, apoptosis induced by active oxygen is easy to generate, the electronic respiratory chain of mitochondria is blocked, and the generation of active oxygen is promoted, so that the tumor cells can be a novel strategy for treating leukemia by using antitumor drugs. Therefore, the establishment of a method for simultaneously detecting and evaluating mitochondrial functions such as mitochondrial active oxygen, mitochondrial autophagy and the like of normal and pathological hematopoietic cells has important significance for research on pathogenesis and clinical treatment of leukemia. Myelodysplastic syndrome (MDS) is a clonal, heterogeneous disease of the blood system, which is characterized by refractory anemia and susceptibility to transformation to leukemia in advanced stages of the disease. Abnormalities associated with mitochondria in patients with MDS are a hotspot in current research of the pathogenesis of MDS. Studies have shown that mitochondrial pathway mediated apoptosis plays an important role in the apoptotic development of MDS, and related studies rely on simultaneous detection and simultaneous assessment of mitochondrial function in hematopoietic cells, including mitochondrial mass, mitochondrial membrane potential.

The invention lays a foundation for discussing the study on malignant diseases of hematopoietic systems such as mitochondrial oxidative respiration and metabolism of leukemia cells, leukemia cell apoptosis mechanism of acute myelogenous leukemia based on mitochondrial dynamics, mitochondrial depolarization-induced lymphoma cell apoptosis and mitochondrial autophagy in the pathogenesis of myelodysplastic syndrome anemia and the like.

The invention is further illustrated below with reference to specific examples, but without limiting the scope of the invention.

Materials and methods

Experimental animal

Obtaining different types of hematopoietic cells from bone marrow of female C57BL/6J (B6) isotype mice (FIG. 1), obtaining MLL-AF9 GFP from bone marrow of MLL-AF9 AML mouse model ⁺ Leukemia cells, FIG. 2 shows hematopoietic cell line granulesFlow chart of the volume study.

Isolation and acquisition of mouse bone marrow cells

B6-Ly5.1 or MLL-AF9 AML mice were sacrificed by cervical removal, sterilized by soaking in medical alcohol for 5min, tibia and femur were dissected from both legs, and placed in 6mm or 10mm petri dishes containing pre-chilled PBS (phosphate buffer saline) solution.

Muscle was removed from the bone using sharp surgical scissors. For each bone, 3ml ice-cold PBE (phosphate buffer electrolyte) was aspirated using a 5 ml syringe and a 25 gauge needle.

The needle was inserted into one end of the bone, and then bone marrow was removed from the small hole and placed into a 5ml tube.

The cell suspension was thoroughly mixed and the cells were placed in a new 5ml tube after passing through a 30-70 μm nylon mesh filter to remove cell clumps.

The number of nucleated cells is counted using a cytometer or an automatic counter. Cells were suspended on ice for use.

Enrichment and purification of Linear negative cells

Whole bone marrow cells were centrifuged to discard supernatant, 80 μl of linear beads were added, incubated on ice for 15min in the dark, washed once with 4ml PBS, centrifuged at 1500rpm/min to discard supernatant, and 12ml PBS to resuspend cells.

The 4 LS sorting columns were rinsed with 3ml PBS respectively, the cell suspension was aliquoted into 4 parts and added to the 4 LS sorting columns respectively, and the collected cell suspension was the Linear negative enriched cell suspension.

Flow type sorting dyeing and feeding machine

Each 50. Mu.l of the staining system contained about 1X 10 ⁷ Individual cells, HSPCs, committed HPCs, CLP monocytes, granulocytes, erythrocytes and lymphocyte antibody combinations (table S2) were added to the cells, respectively, and the cells were incubated on ice for 60-90 minutes. After incubation, 3mL PBS 1500rpm/min was added and centrifuged for 5min, the supernatant was discarded and the cells were washed twice in the same manner. After adding 500 mu LPBS for resuspension, a sample to be tested is prepared by filtering through a nylon net with the diameter of 30-70 mu m. DAPI was added to the final concentration of 1 μg/ml prior to flow-on-machine to exclude dead cells.

Electron microscope detection

The lymphocyte separation liquid separates nucleated cells, and part of the cells are fixed by 2.5% glutaraldehyde for routine ultrastructural observation. The sample was then subjected to osmium acid fixation, dehydration, saturation, and entrapment. The slice thickness is 600nm, the conventional sample slice is dyed with uranium dye liquor for 10min, lead dye liquor for 5-6min, and electron microscope histochemical does not dye. The treated samples were observed using a Hitachi H-600 (Japan) transmission electron microscope, and cell types and maturation stages were analyzed according to cell structures.

Mitochondrial quality detection

Bone marrow cells of 6C 57 mice were divided into A, B, C groups, group A was subjected to Lineare anion-exchange magnet column for detection of HSPC/CLP, committed HPC cells MM; adding erythrocyte lysate into group B for lysis for 2min, and detecting granulocyte/mononuclear and lymphocyte MM; group C was not treated for detection of erythrocyte MM.

The cells of group A were set up in 12 tubes, negative, mitotracker Green Shan Yangguan, FMO control 2 (HSPC/CLP, one control each) and Quan Yang sample 8 (HSPC/CLP, four sub-wells each). The B group cells need to be provided with 12 tubes, namely a negative tube, mitotracker Green Shan Yangguan, an FMO comparison tube 2 tube (one comparison tube for each of granulocyte/monocyte and lymphocyte) and a Quan Yang sample tube 8 tube (four auxiliary holes for each of granulocyte/monocyte and lymphocyte); the C group cells need to be provided with 7 tubes, namely a negative tube, a Mitotracker Green Shan Yangguan tube, an FMO control tube and a full-positive sample tube 4 tube.

Cell counts of each group, sub-packaging and adjusting cell concentration of each tube sample to be 5x10 ⁶ Cells/ml.

HSPC/CLP, committed HPC, granulocyte/monocyte, erythrocyte and lymphocyte antibody combinations (Table S2) were added to the corresponding cell tubes, incubated at 4℃for 30min, 3mL PBS was added, centrifuged at 1500rpm/min for 5min, and the supernatant was discarded.

mu.L of Mitotracker Green working solution with the concentration of 100nM is added to Mitotracker Green Shan Yangguan and Quan Yang sample tubes respectively, incubated at 37 ℃ for 20min in the absence of light, and centrifuged at 1500rpm/min with 3mL of PBS for 5min, and the supernatant is discarded, and the cells are washed twice in the same manner.

After 500 mu LPBS is added for resuspension, a sample to be detected is prepared by filtering through a nylon net with the diameter of 30-70 mu m, 3 mu L of 7-AAD staining solution is added to each tube before the tube is put on a machine, and detection is completed within one hour. All flow templates Mitotracker Green used FITC fluorescence detection channels with photomultiplier voltages kept consistent at 300V.

Mitochondrial membrane potential detection

Bone marrow cells of 6C 57 mice were divided into A, B, C groups, group A was subjected to Lineare anion-exchange magnet column for detection of HSPC/CLP, committed HPC cells MMP; adding erythrocyte lysate into group B for lysis for 2min, and detecting granulocyte/mononuclear cell MMP and lymphocyte MMP; group C was not treated for detection of erythrocyte MMPs.

The cells of group A need to be provided with 10 tubes, namely a negative tube, a Mitosracker Red Shan Yangguan, an FMO control tube 2 tube (HSPC/CLP and a packed HPC control tube respectively), and a full-positive sample tube 6 tube (HSPC/CLP and a packed HPC are respectively provided with three auxiliary holes). The B group of cells are respectively provided with 10 tubes, namely a negative tube, a Mitothecker Red Shan Yangguan, an FMO control tube 2 (one control tube for each of granulocyte/monocyte and lymphocyte) and a whole positive sample tube 6 (three auxiliary holes for each of granulocyte/monocyte and lymphocyte); the group C cells need to be provided with 6 tubes, namely a negative tube, a Mitothecker Red Shan Yangguan tube, an FMO control tube and a full-positive sample tube 3 tube.

Counting cells of each group, subpackaging, and adjusting cell concentration of each tube sample to 5x10 ⁶ Cells/ml.

HSPC/CLP, committed HPC, pellet/monocyte, erythrocyte and lymphocyte antibody combinations (Table S3) were added to the corresponding cell tubes, incubated at 4℃for 30min, 3mL PBS was added, centrifuged at 1500rpm/min for 5min, and the supernatant was discarded.

mu.L of Mitothecker Red working solution with a concentration of 30nM was added to Mitothecker Red Shan Yangguan and Quan Yang sample tubes, respectively, incubated at 37℃for 20min in the absence of light, 3mL of PBS 1500rpm/min was added, centrifuged for 5min, and the supernatant was discarded, and the cells were washed twice in the same manner.

After 500 mu LPBS is added for resuspension, a sample to be detected is prepared by filtering through a nylon net with the diameter of 30-70 mu m, 3 mu L of 7-AAD staining solution is added to each tube before the tube is put on a machine, and detection is completed within one hour. Mitotracker Red in all flow templates used PE fluorescence detection channels, photomultiplier voltages were kept consistent at 390V. The Side Scatter (SSC) voltage needs to be kept consistent at 280V for counting the relative standard deviation (relative standard deviation, rSD) of the SSC signals of various cells.

Leukemia cells and normal cells

Bone marrow cells from 3 MLL-AF9 AML leukemia mice (group A) and three C57 mice (group B) were taken and lysed with red blood cell lysates for 2min, respectively.

The cells of groups A and B were each provided with 6 tubes, negative, mitothecker Red Shan Yangguan, FMO control and full-positive sample tube 3 tubes (three sub-wells).

Two sets of cell counts, sub-packaging and adjusting the cell concentration of each tube sample to be 5x10 ⁶ Cells/ml.

Leukemia stem cell antibody combinations (APC anti c-Kit, APC-Cy7 anti Gr-1) and hematopoietic stem progenitor cell antibody combinations (APC-Cy 7-linear markers, APC anti c-Kit, BV785 anti Sca-1) were added to the corresponding cell tubes, incubated at 4℃for 30min, 3mL PBS was added, centrifuged at 1500rpm/min for 5min, and the supernatant was discarded.

After 500 mu LPBS is added for resuspension, a sample to be detected is prepared by filtering through a nylon net with the diameter of 30-70 mu m, 3 mu L of 7-AAD staining solution is added to each tube before the tube is put on a machine, and detection is completed within one hour.

Mitochondrial active oxygen detection

Bone marrow cells of 6C 57 mice were divided into A, B, C groups, group A was subjected to Lineare anion-exchange magnet column for detection of HSPC/CLP, committed HPC cells ROS; adding erythrocyte lysate into group B for lysing for 2min, and detecting granulocyte/mononuclear and lymphocyte ROS; group C was untreated and used to detect erythrocyte ROS.

The cells of group A need to be provided with 14 tubes, namely a negative tube, ROS Shan Yangguan, FMO reference tube 3 tubes (HSPC, normalized HPC and CLP reference tube respectively), and a full-positive sample tube 9 tubes (HSPC, normalized HPC and CLP sub-wells respectively). The B group of cells need to be provided with 14 tubes, namely a negative tube, an ROS Shan Yangguan tube, an FMO control tube 3 tube (one control tube for granulocytes, monocytes and lymphocytes) and a whole positive sample tube 9 tube (three auxiliary holes for granulocytes, monocytes and lymphocytes); the C group cells need to be provided with 6 tubes, namely a negative tube, an ROS Shan Yangguan tube, an FMO control tube and a full positive sample tube 3 tube.

HSPC, committed HPC, CLP, monocytes, granulocytes, erythrocytes and lymphocytes antibody combinations (Table S4) were added to the corresponding cell tubes, incubated at 4℃for 30min, 3mL PBS was added, centrifuged at 1500rpm/min for 5min, and the supernatant was discarded.

500. Mu.L of Mitosox Red working solution with a concentration of 5. Mu.M was added to ROS Shan Yangguan and Quan Yang sample tubes, respectively, incubated at 37℃for 20min in the absence of light, centrifuged at 1500rpm/min for 5min with 3mL PBS, and the supernatant was discarded, and the cells were washed twice in the same manner.

After 500 mu LPBS is added for resuspension, a sample to be detected is prepared by filtering through a nylon net with the diameter of 30-70 mu m, 3 mu L of 7-AAD staining solution is added to each tube before the tube is put on a machine, and detection is completed within one hour. The MitoSOX Red voltage in all streaming templates needs to be kept consistent.

Leukemia cells and normal cells

mu.L of Mitosox Red working solution with the concentration of 5 mu M is added into Mitosacker Red Shan Yangguan and Quan Yang sample tubes respectively, incubated for 20min at 37 ℃ in the dark, 3mL of PBS 1500rpm/min is added for centrifugation for 5min, the supernatant is discarded, and cells are washed twice in the same way.

After 500 mu LPBS is added for resuspension, a sample to be detected is prepared by filtering through a nylon net with the diameter of 30-70 mu m, 3 mu L of 7-AAD staining solution is added to each tube before the tube is put on a machine, and detection is completed within one hour. All flow templates were subjected to Mitosox Red using a PerCP-Cy5.5 fluorescence detection channel with a photomultiplier voltage of 580V.

Mitochondrial autophagy assay

Bone marrow cells of 6C 57 mice were divided into A, B, C groups, group A was subjected to Lineare anion-exchange magnet column for detection of mitochondrial autophagy of HSPC/CLP, committed HPC cells; adding erythrocyte lysate into group B, and lysing for 2min for detecting mitochondrial autophagy of granulocytes/mononucleated lymphocytes; group C was not treated and used to detect mitochondrial autophagy in erythrocytes.

The cells of group A need to be provided with 12 tubes, namely a negative tube, a Mitophagy Shan Yangguan tube, a FMO control tube 2 tube and a Quan Yang sample tube 8 tube (HSPC/CLP and four auxiliary holes of the assembled HPC respectively). The B group cells need to be provided with 12 tubes, namely a negative tube, a Mitophagy Shan Yangguan tube, an FMO control tube 2 tube and a Quan Yang sample tube 8 tube (four auxiliary holes of granulocyte/monocyte and lymphocyte respectively); the group C cells need to be provided with 7 tubes, namely a negative tube, a Mitophagy Shan Yangguan tube, an FMO control tube and a full-positive sample tube 4 tube.

HSPC/CLP, committed HPC, pellet/monocyte, erythrocyte and lymphocyte antibody combinations (Table S5) were added to the corresponding cell tubes, incubated at 4℃for 30min, 3mL PBS was added, centrifuged at 1500rpm/min for 5min, and the supernatant was discarded.

mu.L of MitophagyDye working solution with the concentration of 100nM is added into Mitophagy Shan Yangguan and Quan Yang sample tubes respectively, incubated at 37 ℃ for 20min in the absence of light, 3mL of PBS 1500rpm/min is added for centrifugation for 5min, the supernatant is discarded, and the cells are washed twice in the same way.

After 500 mu LPBS is added for resuspension, a sample to be detected is prepared by filtering through a nylon net with the diameter of 30-70 mu m, 3 mu L of 7-AAD staining solution is added to each tube before the tube is put on a machine, and detection is completed within one hour. The MitophagyDye voltage in all streaming templates needs to be kept consistent.

Leukemia cells and normal cells

mu.L of Mitophagy working solution with the concentration of 100nM is added into Mitophagy Shan Yangguan and Quan Yang sample tubes respectively, incubated at 37 ℃ for 20min in the absence of light, 3mL of PBS 1500rpm/min is added for centrifugation for 5min, the supernatant is discarded, and the cells are washed twice in the same way.

After 500 mu LPBS is added for resuspension, a sample to be detected is prepared by filtering through a nylon net with the diameter of 30-70 mu m, 3 mu L of 7-AAD staining solution is added to each tube before the tube is put on a machine, and detection is completed within one hour. Mitophagy Dye in all flow templates used PE-Cy5 fluorescence detection channel, photomultiplier voltage was kept consistent at 660V.

Single cell sequencing analysis

Single cell sequencing data of mouse bone marrow hematopoietic stem cells and myeloid progenitor cells were downloaded to NCBI database (GSE 108155, GSE 72857) and analyzed bioinformatically by SeqGeq sequencing analysis software. We analyzed the expression of ten mitochondrial autophagy-related genes in blood cells.

Data analysis

All flow data were obtained by BD AriaIII flow cytometer (BD BiosciAccess, usa) and by FlowJo ^TM v10.6.1 (BD Biosciences, usa) software analysis, flow chart, mean fluorescence intensity per channel (mean) and standard deviation per channel (SD) data were obtained. HSPC tSNE results are obtained by applying a tSNE dimension reduction analysis method, cells with similar protein expression patterns are closely organized together in a t-SNE diagram, and a cell group with similar phenotype is expressed as a highly interconnected node set, so that different cell subgroups are visualized. The final cell classification can be visualized in the form of a color dimension overlaid on the t-SNE map or in the form of a heat map. the proximity of cells in the t-SNE plot reflects their distance in high dimensional space.

Table S1 immunophenotype of various hematopoietic cells

Description: linear markers include B220, ter-119 and Gr-1.

Table S2 flow-sorting antibody combination protocol

Table S3 MitoTracker Green mitochondrial quality inspection antibody combination protocol

Table S4 MitoTracker Red mitochondrial Membrane potential detection antibody combination protocol

Table S5 Mitosox Red mitochondrial reactive oxygen species detection antibody combination protocol

Table S6 Mitophagy detection antibody combination protocol

Table S7 antibody information

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The results of this study demonstrate that HSCs have relatively low mitochondrial metabolic activity, as embodied by lower mitochondrial mass, mitochondrial membrane potential, mitochondrial reactive oxygen species, and relatively high levels of mitochondrial autophagy, as compared to HPC1, HPC2, and committed hematopoietic progenitors (committed HPCs and CLPs). Higher mitochondrial metabolism levels of committed progenitors compared to HSCs may favor differentiation of these progenitors toward the myeloid and the lineage, and myeloid differentiation may require higher levels of aerobic metabolism than the lineage differentiation process. HPC2 shares more stem cell characteristics in terms of mitochondrial number and function than HPC1, committed HPC and CLP. Compared to hematopoietic stem progenitor cells, the mitochondrial metabolism of mature blood cells has a greater heterogeneity, where granulocytes and monocytes present similar metabolic characteristics. Meanwhile, by analyzing autophagy-related genes of hematopoietic stem/progenitor cells, it was initially shown that these cells have heterogeneity in mitochondrial autophagy-related Pink1/Park2, BNIP3/NIX and FUNDC1 pathway selection.

In the study of hematopoietic cells at the same differentiation stage, morphological and functional heterogeneity of hematopoietic cells during differentiation of the myeloid lineage was found to be more pronounced than in the differentiation of the gonococcal lineage. Furthermore, leukemia stem cells (leukemia stem cell, LSC) have higher levels of aerobic metabolism than normal hematopoietic cells, while maintaining stem cell function through high levels of mitochondrial autophagy. The differentiated leukemia cells have higher ROS and MMP levels, and lower levels of mitochondrial autophagy. The research result provides a complete set of methods and basic reference values for systematically researching the mitochondrial metabolism function of different types of hematopoietic cells under physiological and pathological states, and provides standards and references for tumor and aging related research based on mitochondrial metabolism. The specific experimental results are as follows:

morphological diversity of hematopoietic cells at different differentiation stages and heterogeneity of mitochondrial function

FIG. 3a shows the ultrastructural characteristics of mouse bone marrow hematopoietic stem/progenitor cells at different differentiation stages, showing that the nuclei of most hematopoietic cells are irregular and heterochromatin-rich. HSC is minimal in volume, low in cytoplasmic content, and low in mitochondria and other organelles; HPC cytoplasmic content changes are not obvious, and mitophagy is common; CMP has a high cytoplasmic content and an increase in mitochondria and other organelles; GMP is bulky, cytoplasmic and endoplasmic reticulum rich, and mitophagy can be observed; MEP is large in volume, rich in cytoplasm and enlarged in mitochondria; CLP cells are small in size, nuclei are round, and mitochondria are more. It follows that stem progenitor cells, which reside in the initial stages of hematopoietic differentiation, differ in the levels of MM and mitophagy during differentiation of the cell lineage.

FIG. 3b shows the ultrastructural observation of mature cells of the myeloid lineage of the mouse bone marrow at different differentiation stages, from which it can be seen that most mature cells are large in volume, evident in chromatin aggregation, and more cytoplasmic compared to the primitive hematopoietic cells. Granulocyte is cytoplasmic and contains a large number of primary particles; the surface of the Monocyte is provided with tiny protrusions, the cell nucleus is irregular, the cytoplasm is rich, the Monocyte contains vacuoles and a large number of mitochondria, and the particles are fewer; early young erythrocytes in erythroyte have more cytoplasm, regular nuclei and more mitochondria; late stage erythroid nuclei shrink significantly, less mitochondria and other organelles, and enucleation is seen. Ultrastructural differences in myeloid mature cells indicate that there is significant heterogeneity in mitochondrial characteristics of different classes of mature blood cells during hematopoietic differentiation of myeloid.

FIG. 3c is an ultrastructural view of mature cells of the mouse bone marrow lineage at different differentiation stages, from which it can be seen that the different types of lineage cells differ less in volume, chromatin aggregation, less cytoplasm, less mitochondria than CLP, less cytoplasmic granules and other organelles, and less mitophagy phenomenon. Unlike the ultrastructural analysis of hematopoietic stem progenitor cells and myeloid mature cells, the mitochondrial function heterogeneity of the gonococcal mature cells is low, probably due to the close demands of the gonococcal cells in terms of aerobic metabolism and maintenance of cell function. Based on the phenomena observed by ultrastructural technology, characteristics of hematopoietic cell differentiation populations based on flow cell phenotype markers were used for further analysis in order to study the mitochondrial function of hematopoietic cells at different differentiation stages.

Hematopoietic lineage differentiated population characteristics and mitochondrial function heterogeneity

To study the relationship of the differentiated population characteristics of hematopoietic cells to mitochondrial function heterogeneity, a t-distribution neighborhood embedding algorithm (t-distributed Stochastic Neighbor Embedding, tSNE) and a Flow Self-organizing feature map (Flow Self-Organizing feature Map, flowSOM) were used to characterize the population characteristics of hematopoietic cells at different differentiation stages. the tSNE analysis can identify high-dimensional protein marker relatedness, and visualize the high-dimensional similarity of cells, thereby providing a novel method for analyzing flow cell population data. the tSNE analysis reflects the distribution of antigen expression by HSCs, HPCs 1, HPCs 2 and MPPs in HSPC groups, and CMP, GMP and MEPs in committed HPC groups (fig. 4a, fig. 4 d), consistent with the proportion of cell populations obtained from classical flow two-dimensional stepwise loop gates.

As shown in fig. 4b, 4e, HSPC cell populations were divided into 8 groups by FlowSOM, and the heat map color density represents the average expression of a given antigen, normalized to form a heat map. The heat map shows the median intensity of expression of each protein marker for each detected cell population, and clusters are clustered according to differences in protein expression intensity. Live cell flow gates are manually circled on FSC-SSC and DAPI-SSC scatter plots before presenting the data sample set to the FlowSOM. In this study, only cells within the living phylum were extracted and FlowSOM was allowed to further analyze, five surface markers were used in the analysis, and the color of the grid indicated the fluorescent marker intensity. Clusters Pop7, pop6, pop2+ Pop4 and Pop0+ Pop1+ Pop3+ Pop5 allocated according to protein expression in the grid correspond to HSC, MPP, HPC and HPC2 cell populations, respectively, in the classical flow loop gate method. The Committed HPC cell populations were divided into 8 groups by FlowSOM, and the resulting clusters Pop1+Pop2+Pop3+Pop4, pop6 and Pop0+Pop5, respectively, correspond to the CMP, GMP and MEP cell populations in the classical flow loop gate method. The visual representation method of the cell population cluster analysis provided by the FlowSOM as shown in the figures 4c and 4f characterizes the antigen expression distribution condition of the cells represented by the nodes through the node colors and the relative positions among the nodes. The larger the node sector area, the stronger the antigen expression. tSNE and FlowSOM analysis show that hematopoietic stem and progenitor cells can be divided into different characteristic groups based on phenotypic characteristics, and the classification result lays a foundation for researching mitochondrial function heterogeneity of different types of hematopoietic stem and progenitor cells.

Similar to the results of cell surface marker-based hematopoietic stem progenitor cell classification, mouse bone marrow mature hematopoietic cells, including myeloid cells (granulocytes and monocytes), erythroid cells, and gonomic cells (T, B, NK cells), can also be differentiated based on cell phenotype markers to characterize different classes of hematopoietic cells with functional differences. the tSNE analysis results reflect the antigen expression profiles of granulocytes and monocytes (fig. 4 g), erythrocytes (fig. 4 j) and T, B, NK cells (fig. 4 m) of myeloid cells in the mouse bone marrow cell population, consistent with the proportion of cell populations obtained by classical flow two-dimensional stepwise loop gate.

As shown in fig. 4h, 4k and 4n, the myeloid, erythroid and gonococcal cell populations were divided into 8 groups by FlowSOM, the heat map color density represents the average expression of a given antigen, and normalized to form a heat map. The heat map shows the median intensity of expression of each protein marker for each detected cell population, and clusters are clustered according to differences in protein expression intensity. Live cell flow gates are manually circled on FSC-SSC and DAPI-SSC scatter plots before presenting the data sample set to the FlowSOM. In this study, only cells within the living phylum were extracted and FlowSOM was allowed to further analyze, five surface markers were used in the analysis, and the color of the grid indicated the fluorescent marker intensity. In the myeloid cell flow analysis model, clusters Pop6 and Pop4 allocated according to protein expression in the grid correspond to granulocytes and monocytes respectively in the classical flow loop gate method (fig. 4 h). In the erythrocyte flow analysis model, the cluster Pop5 assigned according to protein expression corresponds to the erythrocyte population in the classical flow loop gate method (fig. 4 k). In the flow analysis model of the gonococcal cells, clusters Pop4, pop0, pop2 and Pop1 allocated according to protein expression in the grid correspond to cd4+ T, CD8+ T, B and NK cell populations in the classical flow loop gate method (fig. 4 n). A visual representation of the cell population cluster analysis provided by FlowSOM as shown in fig. 4i, 4l, 4 o. tSNE and FlowSOM analysis show that different types of hematopoietic cells can be classified based on phenotypic antigen characteristics, and functional characteristics of hematopoietic cells at different differentiation stages including mitochondrial function can be studied by taking the classified cell population as a target.

Mitochondrial mass heterogeneity of hematopoietic cells at different differentiation stages

tSNE and FlowSOM analysis demonstrated that hematopoietic cells at different differentiation stages can be divided into different characteristic populations based on phenotypic characteristics, and indices such as MM, MMP, ROS and mitophagy were used for further analysis in order to study mitochondrial function heterogeneity of different differentiation populations. FIG. 5A is a graph showing MM levels of hematopoietic stem progenitor cells, myeloid progenitor cells/gonococcal progenitor cells, and mature blood cells, showing the MM test results of hematopoietic cells at different lineage differentiation stages. During the differentiation of myeloid hematopoietic cells, the MM of primitive hematopoietic stem/progenitor cells (HSCs and MPPs) was lower than that of progenitor cells (HPC 1, HPC2 and committed HPC) and also lower than that of myeloid mature blood cells (granulocytes and monocytes) except erythrocytes, which were lower than that of myeloid hematopoietic cells (fig. 5 Aa). This result reflects that HSCs meet the energy requirements of cells in an anaerobic bone marrow microenvironment, primarily through metabolic means of glycolysis rather than oxidative phosphorylation, so that the MMs of HSCs remain low. This favors HSCs to maintain low ROS levels and maintain self-renewal and multipotent differentiation potential. During the differentiation of the hematopoietic cells of the lineage, the primitive hematopoietic stem and progenitor cells (HSCs and MPPs) were lower in MM than the progenitor cells (HPC 1, HPC2 and CLP), and the mature lymphocytes (T, B, NK cells) were lower in MM than the various hematopoietic stem and progenitor cells (fig. 5 Ab).

In primitive hematopoietic cells, HSCs were significantly lower in MM than progenitor cells (fig. 5Aa, fig. 5 Ab), HPC1 with a tendency for myeloid differentiation was significantly higher in MM than HPC2 with a tendency for gonococcal differentiation (fig. 5 Ac), consistent with differences in MM levels of myeloid committed progenitors (committed HPCs), and of gonococcal committed progenitors (CLPs). Compared to HPC1, committed HPC and CLP, the MM for HPC2 is most similar to HSC. This may suggest that during hematopoietic differentiation, myeloid differentiation requires higher levels of aerobic metabolism than gonococcal differentiation, and HPC2 shares more HSC features in terms of mitochondrial number and function. In mature blood cells, granulocytes and monocytes were higher in MM, cd8+ T and erythrocytes were lower in MM (fig. 5 Ad). The erythrocytes detected in the study were mainly mature erythrocytes, which were energized by anaerobic respiration, and the mitochondria were essentially absent in the cells. Granulocyte and monocyte differentiation is derived from GMP, with similar mitochondrial numbers and functions, as well as several other mitochondrial function assays.

To explore a way to characterize the number of organelles of hematopoietic cells, a simple index of MM characterizing hematopoietic cells, SSC was used for further analysis. SSC represents the granularity of a cell, i.e., the complexity of intracellular organelles, and can potentially reflect the level of variety and number of organelles within a cell. FIG. 5B is a SSC result for hematopoietic cells at different lineage differentiation stages showing SSC numerical levels for hematopoietic stem progenitor cells, myeloid progenitor cells/gonococcal progenitor cells, and mature blood cells. In the process of hematopoietic differentiation of myeloid lineage, primitive hematopoietic stem progenitor cells (HSCs and MPPs) SSC were lower than HPC1, and also lower than myeloid mature blood cells (granulocytes and monocytes) except erythrocytes, which were lower than myeloid lineage class hematopoietic cells, indicating lower levels of primitive and intracellular organelles (fig. 5 Ba). GMP SSC is significantly higher than CMP and MEP, reflecting higher granularity within GMP cells, higher cellular heterogeneity, which may be closely related to its granulocyte differentiation direction. In the process of hematopoietic cell differentiation in the strangles, primitive hematopoietic stem progenitor cells (HSCs and MPPs) SSC are lower than progenitor cells HPC1, but close to HPC2 and CLP. Mature lymphocyte (T, B, NK cells) SSC was lower than various hematopoietic stem and progenitor cells (fig. 5 Bb).

In primitive hematopoietic cells, HSC SSC was higher than MPP, HPC2 and various committed progenitors (CMP, MEP and CLP), but lower than HPC1 and GMP (fig. 5Ba, 5 Bb). HPC1 SSC with a tendency to myeloid differentiation was significantly higher than HPC2 with a tendency to gonococcal differentiation (fig. 5 Bc), but the SSC levels of myeloid committed progenitors (committed HPCs) and gonococcal committed progenitors (CLPs) were relatively close. In mature blood cells, granulocytes and monocytes SSC were higher, B cells and cd8+ T cells SSC were lower (fig. 5 Bd). From the above results, it was found that the SSC level of hematopoietic cells was substantially identical to the MM characteristics, and thus the MM level was reflected well.

Mitochondrial membrane potential level heterogeneity of hematopoietic cells at different differentiation stages

MMP has been further studied in order to investigate the relationship between MM and mitochondrial functions in hematopoietic cells at different differentiation stages. Fig. 5C is a MMP assay of hematopoietic cells at different lineage differentiation stages, showing MMP levels of different types of hematopoietic cells. During the differentiation of myeloid hematopoietic cells, the MMPs of primitive hematopoietic stem and progenitor cells (HSCs and MPPs) were lower than those of progenitor cells (HPC 1, HPC2 and committed HPC) and also lower than that of myeloid mature blood cells (granulocytes and monocytes) except erythrocytes, which were lower than that of myeloid hematopoietic cells (fig. 5 Ca). This result reflects that the growth environment of HSCs is relatively low in oxygen, with few mitochondria in an activated state, and therefore with lower MMPs. During the differentiation of the hematopoietic cells of the lineage, MMPs in primitive hematopoietic stem and progenitor cells (HSCs and MPPs) were lower than those in progenitor cells (HPC 1, HPC2 and CLP), and those in mature lymphocytes (T, B, NK cells) were lower than those in various hematopoietic stem and progenitor cells (fig. 5 Cb). This result may be due to the short life cycle of mature lymphocytes, impaired mitochondrial function leading to mitochondrial depolarization and reduced membrane potential, which in turn leads to altered cell membrane permeability and apoptosis.

In primitive hematopoietic cells, MMPs of HSCs were significantly lower than those of progenitor cells (fig. 5Ca, 5 Cb), those of HPC1 with a tendency to myeloid differentiation were significantly higher than those of HPC2 with a tendency to gonococcal differentiation (fig. 5 Cc), consistent with differences in MMP levels of myeloid committed progenitor cells (committed HPC), and of gonococcal committed progenitor Cells (CLP). Compared to HPC1, committed HPC, HPC2 has the most similar MMP as HSC. In mature blood cells, granulocytes and monocytes had higher MMPs and cd8+ T cells had lower MMPs (fig. 5 CD). From the above results, it can be seen that MMP and MM and SSC levels of hematopoietic cells are substantially identical, and that low levels of MM and impaired mitochondrial function are responsible for lower MMP levels in hematopoietic cells.

Reactive Oxygen Species (ROS) level heterogeneity of hematopoietic cells at different differentiation stages

Fig. 6A is a ROS assay for hematopoietic cells at different lineage differentiation stages, showing ROS levels for different types of hematopoietic cells. During the differentiation of myeloid hematopoietic cells, the ROS content of hematopoietic stem and progenitor cells (HSPCs, HSCs, MPPs, HPCs 1 and HPCs 2) is lower than that of myeloid committed progenitor cells (committed HPCs, CMP, GMPs and MEPs) and also lower than that of mature blood cells (granulocytes, monocytes and erythrocytes). This result reflects that the HSC has a lower MM level and does not energize the cells by oxidative phosphorylation, so that the ROS levels of the cells remain low. The transition of HSCs from resting to proliferative/differentiative states, accompanied by an increase in mTOR activity, leads to an increase in cellular metabolic rate and ROS levels. The rise in ROS levels may be the result of HSCs during proliferation and differentiation to meet higher energy demands. On the other hand, intracellular ROS levels are closely related to HSC differentiation, with higher ROS levels leading to DNA damage of HSCs, which may be one of the mechanisms by which HSCs avoid such damage accumulation. During the differentiation of the hematopoietic cells of the lineage, the ROS content of hematopoietic stem and progenitor cells remains low, followed by mature lymphocytes.

In primitive hematopoietic cells, HSCs had significantly lower ROS levels than progenitor cells (fig. 6Aa, fig. 6 Ab), MEPs had significantly higher ROS levels than CMP, GMP, both myeloid committed progenitors. Consistent with the results of MM and MMPs, the ROS levels of HPC1 with a tendency to myeloid differentiation were higher than those of HPC2 with a tendency to gonococcal differentiation, which also verifies that the myeloid differentiation process requires more mitochondria and thus more ROS. Compared to HPC1, committed HPC, HPC2 has the most similar ROS levels as HSCs, demonstrating that HPC2 shares more of the features of HSCs in terms of mitochondrial numbers and function in hematopoietic stem progenitors from the perspective of aerobic metabolites. In mature blood cells, ROS levels were higher for granulocytes and NK cells, and lower for cd8+ T cells (fig. 6 Ac).

Mitochondrial autophagy level heterogeneity of hematopoietic cells at different differentiation stages

Based on ROS, MM and MMP studies, the mitophagy assay was used for further analysis in order to further explore the mitochondrial metabolism levels of hematopoietic cells at different differentiation stages. Mitophagy selectively eliminates damaged mitochondria to regulate mitochondrial numbers, an effective method for cells to regulate intracellular ROS levels and DNA damage accumulation. FIG. 6B is a mitophagy assay of hematopoietic cells at different lineage differentiation stages showing the mitophagy levels of different types of hematopoietic cells. In the process of myeloid hematopoietic differentiation, the mitophagy level of primitive hematopoietic stem/progenitor cells (HSCs and MPPs) is higher than myeloid mature blood cells (granulocytes, monocytes and erythrocytes) and lower than HPC1 and myeloid progenitor MEPs with a tendency to differentiate myeloid. The mitophagy levels of granulocytes and erythroid lines were lower than those of the various hematopoietic cells of the myeloid lineage (fig. 6 Ba). This result suggests that higher levels of mitophagy in primitive hematopoietic stem and progenitor cells, as compared to mature blood cells, help maintain lower levels of intracellular MMs and ROS, and thus maintain resting state and self-renewal capacity. During the differentiation of the gonococcal cells, the mitophagy levels of primitive hematopoietic stem/progenitor cells (HSC and MPP) were no lower than HPC2 and the gonococcal CLP cells with a tendency to undergo the gonococcal differentiation (fig. 6 Bb).

In primitive hematopoietic cells, the mitophagy level of HSCs was significantly higher than that of myeloid mature blood cells, lower than that of partially committed mature blood cells (fig. 6Ba, fig. 6 Bb), and the mitophagy level of HPC1 with a myeloid differentiation tendency was significantly higher than that of HPC2 with a committed differentiation tendency (fig. 6 Bc), consistent with the difference in mitophagy levels of myeloid committed progenitor cells (committed HPCs), and committed progenitor Cells (CLPs). Compared to HPC2 and CLP, both progenitor cells, HPC1 and committed HPC, closely related to hematopoietic lineage differentiation have relatively higher MM and MMP, and therefore require higher levels of mitophagy to maintain the differentiation capacity of the cells. Compared to HPC1, committed HPC and CLP, the mitophagy ratio of HPC2 is most similar to that of HSC. This result suggests that HPC2 shares more features of HSCs in mitochondrial function in hematopoietic stem and progenitor cells in MM, MMP, ROS and mitophagy, among others. In mature blood cells, B cells had higher mitophagy levels than the various hematopoietic cells and cd8+ T cells had lower mitophagy levels than the various hematopoietic cells (fig. 6Bb, fig. 6 Bd).

Single cell sequencing technology for detecting expression of mitochondrial autophagy related genes in hematopoietic stem progenitor cells

In order to gain an insight into the autophagy-related gene expression procedure of mouse hematopoietic stem/progenitor cells, the results of 10XGenomics single cell gene expression analysis derived from NCBI database were studied for gene expression levels in combination with flow cytometry and electron microscopy techniques. The flow-sorting phenotype of the database-derived hematopoietic stem cells and myeloid progenitor cells was consistent with this experiment (fig. 10a, 10 b). Hematopoietic stem progenitor cells are clustered by bioinformatic integration of these datasets. The functional diversity of myeloid progenitor cells indicates that they have varying degrees of transcriptional specificity for monocytes, granulocytes and erythrocytes. Their transcriptional profile suggests that they are more heterogeneous and subpopulation specific than hematopoietic stem cells. Cluster analysis shows internally significant heterogeneity.

The expression of autophagy-related genes such as Atg7, bcl-2, bcl2l13, drp1, fundc1, nbr1, optn, park2, pink1 and Tax1bp1 were significantly different in different populations of hematopoietic stem cells, different populations of myeloid committed progenitors, and other blood cell populations (fig. 7). Wherein Bcl2l13, nbr1, optn, park2, pink1 and Tax1bp1 are mainly involved in the Pink1/Park2 pathway, bcl-2 is mainly involved in the BNIP3/NIX pathway, drp1 and Fundc1 are mainly involved in the FUNDC1 pathway. This result suggests that the mitophagy heterogeneity of hematopoietic stem and progenitor cells is not only reflected in the number of mitophagy occurrences, but may also be reflected by heterogeneity of the dominant pathways of the different cells that produce mitophagy.

Taking hematopoietic stem cells as an example (FIG. 7a, FIG. 7 b), the Atg7, bcl2, drp1, FUNDC1, nbr1, optn, pink1 and Tax1bp1 genes were all expressed in three HSC populations, but Drp of Pop2, pop3, tax1bp1 genes were significantly more expressed than the other two populations, respectively, and Park2 genes were less expressed in Pop1, suggesting that there might be different mitophagy dominant pathways in the three HSC populations. Tax1bp1 as autophagy receptor ubiquitin substrate was attached to autophagosome membrane during the process of selective autophagy clearance of damaged mitochondria. Pink1 is a serine/threonine kinase that is able to specifically localize on depolarized mitochondria, activate Parkin by phosphorylating ubiquitin, and induce mitophagy by recruiting autophagy receptors. Drp1 mediates mitochondrial division and mitophagy primarily through the FUNDC1 pathway. This result indicates that the Pink1/Park2, BNIP3/NIX and FUNDC1 pathways are ubiquitous in the HSC mitochondrial autophagy process, and the Pink1/Park2 and FUNDC1 pathways are simultaneously active in Pop2 and Pop3 populations to recruit autophagy receptors such as Drp1 and Tax1bp1 to participate in the mitophagy process. Pop1 is a possible dominant pathway with both FUNDC1 and BNIP 3/NIX. The heat map (FIG. 7 c) shows the same gene expression profile and shows that Pop2 and Pop3 have a denser autophagy-related gene expression than Pop 1.

In the committed progenitor cell population (FIG. 7d, FIG. 7 e), the committed HPC was determined to contain three cell populations GMP, MEP and CMP by recognition of the expression of the characteristic genes CD34 and CD 16/32. Atg7, drp1, FUNDC1, nb1, pink1 and Tax1bp1 genes are all expressed and distributed in three committed HPC groups, but Bcl2l13 and Park2 of MEP, the gene expression of Bcl2 of CMP is respectively obviously stronger than that of other two groups, and Park2 of GMP and Bcl2 of MEP are expressed less, which means that MEP takes Pink1/Park2 and FUNDC1 as dominant pathways, CMP takes BNIP3/NIX pathway as dominant pathway, and GMP takes FUNDC1 as possible dominant pathway. The heat map (fig. 7 f) shows the same results and shows that MEP and CMP have a denser autophagy-related gene expression compared to GMP.

The heat map (hetmap) comprehensively reflects the expression level of the gene or protein, the distribution of the cell subsets expressing the marker gene, and the number of cells expressing the marker gene in the cell subsets, and is the main way to perform the expression of the marker gene.

In order to explore the functional heterogeneity of hematopoietic cells at the same differentiation stage based on cell surface marker classification, ultrastructural and flow cytometry analyses were used for further investigation, on the basis of studies of mitochondrial function of hematopoietic cells at different differentiation stages. From electron microscopic observation of the same field of view of hematopoietic cells classified according to the same phenotype, it was found that HPC (fig. 8 Aa), MEP (fig. 8 Ab), GMP (fig. 8 Ac) and eryhyocyte (fig. 8 Ad) were different in cell volume size, and that the difference in cytoplasmic content and the number of mitochondria was large, indicating that there was a large heterogeneity in mitochondrial function inside these several types of myeloid hematopoietic cells. However, the morphological differences between the mature lymphocyte CD4+T (FIG. 8 Ae) and B (FIG. 8 Af) cells were not apparent, showing lower mitochondrial function heterogeneity.

To more completely explore mitochondrial function of hematopoietic cells at the same differentiation stage, SSC-based rSD was used to evaluate the degree of difference between the components within one cell population. Figures 8Ba, 8Bb show rSD levels of hematopoietic stem progenitor cells, myeloid committed progenitor cells/gonomic committed progenitor cells, and mature blood cell populations, respectively, differentiated by lineage. During differentiation of myeloid hematopoietic cells, the rSD of the primitive hematopoietic stem/progenitor (HSC and MPP) population was lower than progenitor (HPC 1, HPC2 and committed HPC) and also lower than the various myeloid mature blood cells (granulocytes, monocytes and erythrocytes), and the erythroid population rSD was higher than the myeloid hematopoietic cells (fig. 8 Ba). The smaller SSCrSD of HSC and MPP indicates that the SSC distribution within the primordial cell population deviates less from the mean value and has lower heterogeneity relative to the more mature cells. rSD of HPC1, MEP, GMP and eryhyocyte were higher reflecting higher heterogeneity of cell structure and function, consistent with ultrastructural observations. During the differentiation of the hematopoietic cells of the lineage, the primary hematopoietic stem and progenitor cell (HSC and MPP) population rSD was lower than the progenitor cells (HPC 1, HPC2 and CLP), the mature lymphocyte (T, B, NK cell) population rSD was lower than the various hematopoietic stem and progenitor cells (fig. 8 Bb), reflecting their lower heterogeneity in cell composition and structure, consistent with the results observed for ultrastructural. The rSD changes of different types of hematopoietic cells are consistent with their MM and MMP characteristics.

In primitive hematopoietic cells, rSD of HSC populations was significantly lower than progenitor cells (fig. 8Ba, fig. 8 Bb), rSD of populations of HPC1 with a tendency for myeloid differentiation was significantly higher than HPC2 with a tendency for gonococcal differentiation (fig. 8 Bc), consistent with MMP differences in myeloid committed progenitor cells (committed HPC), and in gonococcal committed progenitor Cells (CLP). In mature blood cells, the red blood cell population rSD was higher and the gonococcal cell population rSD was lower (fig. 8 Bd). Morphological diversity and mitochondrial function heterogeneity of hematopoietic cells at the same stage of differentiation suggest that cellular phenotypic markers have limitations in characterizing hematopoietic cell function, and morphological and functional heterogeneity among cells within a population of cells with the same cellular phenotypic markers, indicating that studies targeting a population of cells may reveal functional characteristics of a class of cells better than single cell studies. Therefore, the indexes such as rSD and the like and the electron microscope technology provide ideas and methods for researching the mitochondrial function of hematopoietic cells at the same differentiation stage.

Mitochondrial function heterogeneity of pathological versus normal hematopoietic cells

FIGS. 9a-c are mitochondrial function assays of bone marrow pathological cells and normal mouse hematopoietic cells of mice in the acute myeloid leukemia model, reflecting ROS, MMP, and mitophagy levels of LSC, non-LSC, LSK, and non-LSK, respectively. The results show that LSC has high ROS, MMP and mitophagy levels compared to non-LSC and has statistical differences. LSC has high ROS, MMP, and mitophagy levels compared to LSK, and has significant statistical differences. This result suggests that LSC has higher levels of aerobic metabolism while maintaining stem cell function by clearing excess mitochondria through high levels of mitophagy. Compared to non-LSK, non-LSC has a high ROS, MMP level, and a low mitophagy level, and has significant statistical differences. This phenomenon suggests that mitochondria in non-LSCs are prone to accumulation, while weaker mitophagy and higher intracellular ROS levels are detrimental to maintenance of cellular function and are prone to triggering apoptosis. Compared to non-LSK, LSK has a lower ROS, mitophagy level, while MMP is higher and has very significant statistical differences. This phenomenon indicates that less harmful substances such as ROS accumulate in LSK and that cell function can be maintained by lower levels of mitophagy.

Claims

1. A method for detecting mitochondrial function of hematopoietic cells, characterized by: the method comprises the following steps: simultaneously detecting and evaluating Mitochondrial Mass (MM), mitochondrial Membrane Potential (MMP), mitochondrial Reactive Oxygen Species (ROS), and mitochondrial autophagy levels of normal hematopoietic cells and pathological hematopoietic cells including hematopoietic stem cells, hematopoietic progenitor cells, and mature blood cells; the normal hematopoietic cells are divided into myeloid hematopoietic cells and gonococcal hematopoietic cells during differentiation; the pathological hematopoietic cells comprise Leukemia Stem Cells (LSC) and leukemia cells (non LSC), and the pathological cells are normal primitive hematopoietic cells (LSK) and normal mature hematopoietic cells (non LSK) in contrast; the simultaneous detection includes: the same flow cytometry is adopted, the same time point is used for detection, and the incubation time of fluorescent Dye is the same, namely, the mitochondrial mass Mitotracker Green, mitochondrial membrane potential Mitothecker Red, mitochondrial active oxygen Mitosox Red and mitochondrial autophagy Mitophagy Dye are respectively incubated for 20-30min at 37 ℃ in a dark place; the voltage of a photomultiplier tube for detecting fluorescent dye by mitochondrial function is respectively consistent in each flow template;

the simultaneous evaluation includes: the flow JoTM v10.6.1 software is used for obtaining flow data and subtracting the spectrum overlapping interference among all fluorescence channels according to the multi-parameter fluorescence compensation adjustment principle to obtain flow chart, average fluorescence intensity (mean) of all channels and Standard Deviation (SD) data of all channels; obtaining a HSPC tSNE result by applying a tSNE dimension reduction analysis method, wherein cells with similar protein expression patterns are closely organized together in a t-SNE diagram, and a cell group with similar phenotype is expressed as a highly interconnected node set, so that different cell subgroups are visualized; the final cell classification is visualized in the form of a color dimension overlaid on the t-SNE map or in the form of a heat map; the proximity of cells in the t-SNE plot reflects their distance in high dimensional space;

Based on the detection result of the MM,

during myeloid hematopoietic cell differentiation, HSCs and MPPs have MMs below HPC1, HPC2 and committed HPCs, and also below granulocytes and monocytes, erythrocytes have MMs below HSC, MPP, HPC1, HPC2, committed HPCs, granulocytes and monocytes;

during the differentiation of the hematopoietic cells of the lineage, HSCs and MPPs had MMs below HPC1, HPC2 and CLP, T, B, NK cells had MMs below HSC, MPP, HPC, HPC2 and CLP;

in primitive hematopoietic cells, HSCs have a lower MM than progenitor cells, HPC1 with a higher tendency for myeloid differentiation than HPC2 with a tendency for gonococcal differentiation;

compared to HPC1, committed HPC and CLP, the MM for HPC2 is most similar to HSC;

in mature blood cells, the MMs of granulocytes and monocytes are higher, and those of cd8+ T and erythrocytes are lower;

based on the results of the MMP assay,

during myeloid hematopoietic cell differentiation, HSCs and MPPs were lower than HPC1, HPC2 and committed HPC, and also lower than granulocytes and monocytes, erythrocytes MMPs were lower than HSC, MPP, HPC, HPC2, CMP, MEP, GMP, granulocytes and monocytes;

during the differentiation of the hematopoietic cells of the lineage, MMPs in HSCs and MPPs were lower than HPC1, HPC2 and CLP, and MMPs in T, B, NK cells were lower than HSC, MPP, HPC, HPC2 and CLP;

In primitive hematopoietic cells, MMPs of HSCs are lower than progenitor cells, MMPs of HPC1 with a tendency for myeloid differentiation are higher than HPC2 with a tendency for gonococcal differentiation;

compared to HPC1, committed HPC, HPC2 has the most similar MMP as HSC;

in mature blood cells, granulocytes and monocytes have higher MMPs and cd8+ T cells have lower MMPs;

based on the ROS detection result,

during myeloid hematopoietic cell differentiation, the ROS content of HSPC, HSC, MPP, HPC1 and HPC2 is lower than that of committed HPC, CMP, GMP and MEP, and also lower than granulocytes, monocytes and erythrocytes;

in the process of differentiation of the hematopoietic cells of the stranguria system, the ROS content of the hematopoietic stem progenitor cells is still in a low position, and the hematopoietic stem progenitor cells are mature lymphocytes;

in primitive hematopoietic cells, HSCs have ROS levels lower than progenitor cells, MEPs have ROS levels higher than CMP, GMP, both myeloid progenitor cells;

HPC1 with a tendency to myeloid differentiation has higher ROS levels than HPC2 with a tendency to gonococcal differentiation;

compared to HPC1, committed HPC, HPC2 has the most similar ROS levels as HSCs;

in mature blood cells, granulocytes and NK cells have higher ROS levels, cd8+ T cells have lower ROS levels;

based on the results of the detection of the level of autophagy in mitochondria,

during myeloid hematopoietic differentiation, the autophagy levels of HSCs and MPPs are higher than granulocytes, monocytes and erythrocytes, lower than HPC1 and myeloid progenitor MEPs with myeloid differentiation tendencies, and lower than HSC, MPP, HPC1, HPC2, CMP, MEP, GMP and monocytes;

During the differentiation of the gonococcal cells, the autophagy level of HSC and MPP is not lower than HPC2 and the gonococcal progenitor CLP, which have a tendency to be gonococcal;

in primitive hematopoietic cells, HSCs have higher autophagy levels than granulocytes, monocytes and erythrocytes, lower than cd4+ T cells and B cells, and HPC1 with a tendency for myeloid differentiation has higher autophagy levels than HPC2 with a tendency for lineage differentiation;

compared to HPC1, committed HPC and CLP, the autophagy ratio of HPC2 is most similar to HSC;

in mature blood cells, the autophagy level of B cells is higher than HSC, MPP, HPC1, HPC2, CLP, cd4+ T cells, cd8+ T cells, NK cells, the autophagy level of cd8+ T cells is lower than HSC, MPP, HPC1, HPC2, CLP, cd4+ T cells, B cells, NK cells;

based on the results of the detection of the pathological hematopoietic cells and the normal hematopoietic cells,

ROS, MMP, and autophagy levels of LSC are higher compared to non-LSC; ROS, MMP, and autophagy levels of LSC are higher compared to LSK; compared with non-LSK, the level of ROS and MMP of non-LSC is higher, and the autophagy level is lower; compared to non-LSK, LSK has a lower ROS, autophagy level, and MMP is higher.

2. The method for detecting mitochondrial function of hematopoietic cells according to claim 1, wherein: the photomultiplier voltage of the mitochondrial mass function detection fluorescent dye Mitotracker Green is kept consistent at 300V in each flow template.

3. The method for detecting mitochondrial function of hematopoietic cells according to claim 1, wherein: the voltage of a photomultiplier of the fluorescent dye Mitosacker Red detected by the mitochondrial membrane potential function is respectively kept to be identical to 390V in each flow template.

4. The method for detecting mitochondrial function of hematopoietic cells according to claim 1, wherein: the photomultiplier voltage of the mitochondrial active oxygen function detection fluorescent dye Mitosox Red is kept consistent to 580V in each flow template.

5. The method for detecting mitochondrial function of hematopoietic cells according to claim 1, wherein: the photomultiplier voltages of the mitochondrial autophagy function detection fluorescent Dye Mitophagy Dye are respectively kept consistent to be 660V in each flow template.