Patent Application
20230027247
The present application relates to the field of biomedicine, particularly to a small molecule compound for expanding hematopoietic stem cells (HSCs), more particularly to a small molecule inhibitor of cell signaling pathway, a composition thereof, and use of the small molecule inhibitor and the composition thereof in expanding hematopoietic stem cells.
Hematopoietic stem cells (HSCs) are a group of heterogeneous primitive hematopoietic cells in the blood system, with two important characteristics of self-renewal and multi-lineage differentiation. When the body is in a healthy state, HSCs in the body are in a quiescent state for a long time. When the body is in a state of disease or severe blood loss, HSCs are activated and enter a state of self-renewal and multi-lineage differentiation to maintain blood system stability and body homeostasis. The self-renewal property of HSCs is beneficial to maintain the stemness of progeny HSCs, while the multi-lineage differentiation property of HSCs allows them to differentiate into a variety of mature blood cells, such as myeloid cells (granulocytes, monocytes, erythrocytes and platelets), lymphoid cells (T cells and B cells). The properties of HSCs facilitate the differentiation of HSCs when the body needs them.
These properties of HSCs make it possible to treat hematological diseases by hematopoietic stem cell transplantation (HSCT). HSCs include long term hematopoietic stem cells (LT-HSCs) and short term hematopoietic stem cells (ST-HSCs). The former has high self-renewal ability and can carry out hematopoietic reconstruction throughout the life cycle of the body; while the latter can only maintain the function of hematopoietic reconstruction for a limited time. In 1959, Thomas et al. used bone marrow hematopoietic stem cells for the first hematopoietic stem cell transplantation in human history, clinically treating leukemia to restore normal hematopoietic function in a patient. In the following decades, through the continuous efforts of scientific researchers, hematopoietic stem cell transplantation has not only been used to treat a variety of blood system diseases, but also used to treat immunodeficiency diseases and neurodegenerative diseases.
At present, there are three main sources of HSCs, i.e., bone marrow (BM), mobilized peripheral blood (mPB), and umbilical cord blood (CB). The collection of bone marrow hematopoietic stem cells is invasive and insufficient, so this method is basically eliminated. The proportion of HSCs in human peripheral blood is very low (less than 0.1%), and it is necessary to use granulocyte colony stimulating factor (G-CSF) to mobilize hematopoietic stem cells to migrate from bone marrow to peripheral blood for transplantation. In clinical applications, the mobilization effect is often ineffective, and the number of HSCs contained in peripheral blood is insufficient, resulting in repeated mobilizations or transplantation failure. Moreover, the HSCs collected by these two methods all require human leukocyte antigen (HLA) matching between the donor and the patient. HLA matching is difficult, and once mismatch occurs, it will lead to graft-versus-host reaction (GVHD), and a patient with GVHD will die from immune system disorders.
As a new source of HSCs, umbilical cord blood has many advantages. Firstly, umbilical cord blood HSCs have low requirements for HLA matching, allowing partial HLA mismatch, and the incidence of GVHD after transplantation is low, thereby alleviating the difficulty of traditional HSCT matching; secondly, umbilical cord blood collection is convenient, harmless to the donor and has no ethical issues, and HSCs have strong hematopoietic ability. These advantages make umbilical cord blood a preferred source of HSCs for treating diseases in future.
However, due to the small amount of cells in a single collection of umbilical cord blood, the overall number of HSCs is small, so it is limited to children or light-weight adults for HSCs transplantation, and cannot meet the transplant needs of larger-weight adults. When the number of transplanted HSCs is insufficient, neutrophil recovery is delayed in a patient, leading to an increased risk of GVHD. These have become bottlenecks restricting the clinical application of umbilical cord blood.
In conclusion, the safety and efficacy of hematopoietic stem cell transplantation depend on the content of transplanted HSCs. If the number of HSCs can be expanded in vitro, the success rate of hematopoietic stem cell transplantation can be improved.
Researchers continue to work hard and try to explore different methods to achieve in vitro expansion of hematopoietic stem cells. One of the strategies for in vitro expansion of HSCs is to utilize small-molecule compounds (SMCs) targeting HSCs. SMCs are readily available in source, easy to mass produce, stable in nature, clear in structure, and easy to control in concentration, and have been widely used in medical research. In the current HSCs in vitro expansion technology, SMCs can significantly increase the expansion folds of HSCs. For example, the AhR aryl hydrocarbon receptor inhibitor StemRegenin1 (SR1) is the first screened SMC that can expand HSCs in vitro. The pyrimidine indole derivative UM171 is also able to expand HSCs in vitro, but it does not act through the AhR cell signaling pathway. Transcriptome analysis shows that, UM171 does not down-regulate AhR cell signaling pathway, but inhibits erythrocyte and megakaryocyte differentiation-related genes. The combination of the two can increase the amplification folds of HSCs.
Histone deacetylases (HDACs) are known cell signaling pathways. Histones can regulate the transcriptional process of specific genes, cell proliferation and differentiation through acetylation or deacetylation. Existing research results reveal that, histone acetylation plays a role in the self-renewal and proliferation of HSCs. HDAC inhibitors TSA, trapoxin and chlamydocin, can regulate histone acetylation in vitro to promote the self-renewal and proliferation of HSCs.
Src is encoded by the Src proto-oncogene, and is a non-receptor protein kinase with tyrosine protein kinase activity. It exists in the cytoplasm, and can be activated by a variety of cell surface receptors to participate in mediating multiple cell signaling pathways, thereby regulating cell proliferation, differentiation and other processes, and it is a key molecule in multiple cell signaling pathways. For example, after Src is activated, it cooperates with p52Shc to activate the mitogen-activated protein kinase (MAPK) cell signaling pathway, and participates in the downstream of MAPK to regulate cell growth and differentiation process; Src can also activate the STAT cell signaling pathway and promote the transcription of related genes. So far, no studies have reported that Src inhibitors help to maintain the sternness of HSCs during in vitro expansion, but in the study of the present invention we found that Src inhibitors can promote the proliferation of HSCs and maintain the sternness of HSCs.
Although it has been found that small molecule inhibitors of some cell signaling pathways can promote the proliferation of HSCs, in this field it still needs to continue to search for small molecule compounds that can promote the proliferation of HSCs more effectively and can maintain the sternness of the proliferated HSCs, and a comprehensive strategy to achieve this purpose, so as to meet the clinical needs.
The problem to be solved by this application is to screen out the best small molecule compounds and their compositions that can promote the in vitro expansion of HSCs while maintaining a high proportion of sternness of HSCs by studying the key factors that regulate cell signaling pathways, so as to solve the problem in the prior art that the number of HSCs expanded in vitro is still insufficient.
Firstly, the applicant's research found that multiple small-molecule inhibitors targeting Src in the STAT cell signaling pathway can well maintain the sternness of HSCs in vitro culture. Src targets play an important role in the expansion of hematopoietic stem cells in the STAT cell signaling pathway, while maintaining the sternness of hematopoietic stem cells, which has not been reported in previous studies.
Further, the applicant found that during the in vitro culture of HSCs, the combinations of HDAC inhibitors, such as SAHA and Valproic acid (VPA), with small molecule inhibitors targeting Src can well maintain the sternness of HSCs, and its effect is far better than SR1 and UM171 which have been found in the prior art.
Therefore, in one aspect the present application finds that small molecule inhibitors of the cell signaling pathway of signal transducer and activator of transcription (STAT), such as a small molecule inhibitor targeting Src target, can promote the proliferation of HSCs and maintain the sternness characteristics of HSCs. In some embodiments, the present application provides a method for promoting the proliferation of HSCs and maintaining the sternness of HSCs, comprising: in vitro contacting the HSCs with a culture medium containing a small molecule inhibitor of the STAT cell signaling pathway or a small molecule inhibitor of other cell signaling pathway than the STAT cell signaling pathway. In some embodiments, the small molecule inhibitor of the STAT cell signaling pathway is a small molecule inhibitor targeting Src. In some embodiments, the small molecule inhibitor targeting Src is one or more selected from the group consisting of: Dasatinib, Quercetin, UM-164, KX2-391, and KX1-004. In some embodiments, the small molecule inhibitor targeting Src is one or more selected from the group consisting of: Dasatinib, UM-164, and KX1-004.
In some embodiments, the small molecule inhibitor targeting Src is used in combination with the small molecule inhibitor of the other cell signaling pathway. In some embodiments, the small molecule inhibitor of the other cell signaling pathway is one or two or more selected from the group consisting of: a small molecule inhibitor targeting HDAC, a small molecule inhibitor targeting PKC, and a small molecule inhibitor targeting JNK. In some embodiments, the small molecule inhibitor targeting Src is used in combination with a small molecule inhibitor targeting HDAC, a small molecule inhibitor targeting PKC, or a small molecule inhibitor targeting JNK. In some embodiments, the small molecule inhibitor targeting Src is used in combination with a small molecule inhibitor VPA targeting HDAC, a small molecule inhibitor SAHA targeting HDAC, a small molecule inhibitor Enzastaurin targeting PKC, or a small molecule inhibitor JNK-IN-8 targeting JNK. In some embodiments, the small molecule inhibitor Dasatinib targeting Src is used in combination with VPA, or SAHA. In some embodiments, the small molecule inhibitor of the cell signaling pathway of signal transducer and activator of transcription (STAT), e.g., a small molecule inhibitor targeting Src, such as Dasatinib, UM-164 or KX1-004, maintains the HSCs with a CD34+CD45+CD90+CD45RA-CD38− phenotype accounting for more than 8%, 10%, 15%, 20%, 25%, or 30% of all cells; for example, maintains HSCs with a CD34+CD45+CD90+CD45RA-CD38− phenotype accounting for 5%-35%, 10%-35%, 15%-35%, 20%-35%, 25%-35%, 5%-30%, 10%-30%, 20%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, 15%-20% of all cells; maintains CD34+ cells accounting for more than 65%, 70%, 75%, 80%, or 85% of all cells.
In some embodiments, the present application provides a composition for maintaining the sternness of HSCs, comprising a small molecule inhibitor of the STAT cell signaling pathway. In some embodiments, the small molecule inhibitor of the STAT cell signaling pathway is a small molecule inhibitor targeting Src. In some embodiments, the small molecule inhibitor targeting Src is one or more selected from the group consisting of: Dasatinib, Quercetin, UM-164, KX2-391, and KX1-004. In some embodiments, the composition further comprises the small molecule inhibitor of the other cell signaling pathway. In some embodiments, the small molecule inhibitor of the other cell signaling pathways comprises a small molecule inhibitor targeting HDAC, a small molecule inhibitor targeting PKC, or a small molecule inhibitor targeting JNK. In some embodiments, the small molecule inhibitor of the other cell signaling pathway comprises a small molecule inhibitor VPA targeting HDAC, a small molecule inhibitor SAHA targeting HDAC, a small molecule inhibitor Enzastaurin targeting PKC, or a small molecule inhibitor JNK-IN-8 targeting JNK. In some embodiments, the composition further comprises SFEMII medium, growth factor Flt-3L, growth factor SCF, growth factor TPO, and growth factor IL-6. In some embodiments, the composition consists of a small molecule inhibitor of STAT cell signaling pathway and/or a small molecule inhibitor of the other cell signaling pathway, SFEMII medium, growth factor Flt-3L, growth factor SCF, growth factor TPO, and growth factor IL-6. In some embodiments, the composition maintains HSCs with a CD34+CD45+CD90+CD45RA-CD38− phenotype accounting for more than 8%, 10%, 15%, 20%, 25%, or 30% of all cells, for example, maintains HSCs with CD34+CD45+CD90+CD45RA-CD38− phenotype accounting for 5%-35%, 10%-35%, 15%-35%, 20%-35%, 25%-35%, 5%-30%, 10%-30%, 20%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, or 15%-20% of all cells. In some embodiments, the composition maintains CD34+ cells accounting for more than 65%, 70%, 75%, 80%, or 85% of all cells.
In some embodiments, the small molecule inhibitor of the cell signaling pathway of signal transducer and activator of transcription (STAT), e.g., a small molecule inhibitor targeting Src, such as Dasatinib, UM-164, or KX1-004, is used in combination with the small molecule inhibitor of the other cell signaling pathway, such as VPA or SAHA, to maintain HSCs with a CD34+CD45+CD90+CD45RA-CD38− phenotype accounting for more than 8%, 10%, 15%, 20%, 25%, or 30% of all cells; for example, to maintain HSCs with a CD34+CD45+CD90+CD45RA-CD38− phenotype accounting for 5%-35%, 10%-35%, 15%-35%, 20%-35%, 25%-35%, 5%-30%, 10%-30%, 20%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, or 15%-20% of all cells; to maintain CD34+ cells accounting for more than 65%, 70%, 75%, 80%, or 85% of all cells. In some embodiments, the concentration of each inhibitor in the culture medium is:
Dasatinib: 0.1 μM-50 μM, preferably 0.5 μM-40 μM, more preferably 0.5 μM-30 μM, most preferably 0.5 μM-10 μM;
SAHA: 10 nM-20 μM, preferably 20 nM-15 μM, more preferably 30 nM-10 μM, most preferably 0.1 μM-10 μM;
VPA: 10 μM-2000 μM, preferably 10 μM-1500 μM, more preferably 10 μM-1000 μM, most preferably 100 μM-1000 μM;
JNK-IN-8: 0.1 μM-20 μM, preferably 0.5 μM-15 μM, more preferably 0.5 μM-10 μM, most preferably 1 μM-10 μM;
EPZ004777: 0.1 μM-50 μM, preferably 0.5 μM-40 μM, more preferably 0.5 μM-30 μM, most preferably 0.5 μM-10 μM;
DZNeP: 1 nM-500 nM, preferably 5 nM-400 nM, more preferably 10 nM-300 nM, most preferably 10 nM-250 nM;
UM-164: 0.1 μM-1000 μM, preferably 0.5 μM-500 μM, more preferably 1 μM-100 μM, most preferably 1 μM-10 μM;
KX2-391: 0.1 nM-1000 nM, preferably 1 nM-1000 nM, more preferably 10 nM-500 nM, most preferably 10 nM-100 nM;
KX1-004: 0.1 μM-1000 μM, preferably 1 μM-1000 μM, more preferably 10 μM-500 μM, most preferably 10 μM-100 μM.
In one aspect, the present application provides a method for promoting the proliferation of HSCs and maintaining the sternness of the HSCs, comprising: in vitro contacting the HSCs with a culture medium containing one or more small molecule inhibitors selected from the group consisting of: 1) a small molecule inhibitor VPA targeting HDAC; 2) a small molecule inhibitor SAHA targeting HDAC; 3) a small molecule inhibitor Enzastaurin targeting PKC; and 4) a small molecule inhibitor JNK-IN-8 targeting JNK.
In one aspect, the present application provides a composition for maintaining the sternness of HSCs, wherein the composition comprises any combination selected from the group consisting of: SAHA+EPZ004777, SAHA+DZNeP, SAHA+Dasatinib, VPA+Dasatinib, SAHA+JNK-IN-8, or SAHA+VPA. In some embodiments, the composition further comprises SFEMII medium, growth factor Flt-3L, growth factor SCF, growth factor TPO, and growth factor IL-6. In some embodiments, the composition consists of: any combination selected from SAHA+EPZ004777, SAHA+DZNeP, SAHA+Dasatinib, VPA+Dasatinib, SAHA+JNK-IN-8 or SAHA+VPA, and SFEMII medium, growth Factor Flt-3L, growth factor SCF, growth factor TPO, and growth factor IL-6. In some embodiments, the composition for maintaining the sternness of HSCs may also facilitate to maintain the proportion of CD34+ cells. In some embodiments, the composition maintains HSCs with a CD34+CD45+CD90+CD45RA-CD38− phenotype accounting for more than 8%, 10%, 15%, 20%, 25%, or 30% of all cells, for example, maintains HSCs with CD34+CD45+CD90+CD45RA-CD38-phenotype accounting for 5%-35%, 10%-35%, 15%-35%, 20%-35%, 25%-35%, 5%-30%, 10%-30%, 20%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, or 15%-20% of all cells; maintains CD34+ cells accounting for more than 65%, 70%, 75%, 80%, or 85% of all cells. In some embodiments, the concentration of each inhibitor in the culture medium is:
Dasatinib: 0.1 μM-50 μM, preferably 0.5 μM-40 μM, more preferably 0.5 μM-30 μM, most preferably 0.5 μM-10 μM;
SAHA: 10 nM-20 μM, preferably 20 nM-15 μM, more preferably 30 nM-10 μM, most preferably 0.104-10 μM;
VPA: 10 μM-2000 μM, preferably 10 μM-1500 μM, more preferably 10 μM-1000 μM, most preferably 100 μM-1000 μM;
JNK-IN-8: 0.1 μM-20 μM, preferably 0.5 μM-15 μM, more preferably 0.5 μM-10 μM, most preferably 1 μM-10 μM;
EPZ004777: 0.1 μM-50 μM, preferably 0.5 μM-40 μM, more preferably 0.504-30 μM, most preferably 0.5 μM-10 μM;
DZNeP: 1 nM-500 nM, preferably 5 nM-400 nM, more preferably 10 nM-300 nM, most preferably 10 nM-250 nM.
In one aspect, the present application provides a composition for maintaining the sternness of HSCs, wherein the composition comprises any combination selected from: SAHA+EPZ004777+DZNeP, or SAHA+VPA+Dasatinib. In some embodiments, the composition further comprises SFEMII medium, growth factor Flt-3L, growth factor SCF, growth factor TPO, and growth factor IL-6. In some embodiments, the composition consists of: SAHA+EPZ004777+DZNeP or SAHA+VPA+Dasatinib, and SFEMII medium, growth Factor Flt-3L, growth factor SCF, growth factor TPO, and growth factor IL-6. In some embodiments, the composition maintains HSCs with a CD34+CD45+CD90+CD45RA-CD38− phenotype accounting for more than 8%, 10%, 15%, 20%, 25%, or 30% of all cells, for example, maintains HSCs with CD34+CD45+CD90+CD45RA-CD38− phenotype accounting for 5%-35%, 10%-35%, 15%-35%, 20%-35%, 25%-35%, 5%-30%, 10%-30%, 20%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, or 15%-20% of all cells; maintains CD34+ cells accounting for more than 65%, 70%, 75%, 80%, or 85% of all cells. In some embodiments, the concentration of each inhibitor in the culture medium is:
Dasatinib: 0.1 μM-50 μM, preferably 0.5 μM-40 μM, more preferably 0.5 μM-30 μM, most preferably 0.5 μM-10 μM;
SAHA: 10 nM-20 μM, preferably 20 nM-15 μM, more preferably 30 nM-10 μM, most preferably 0.104-10 μM;
VPA: 10 μM-2000 μM, preferably 10 μM-1500 μM, more preferably 10 μM-1000 μM, most preferably 100 μM-1000 μM;
EPZ004777: 0.1 μM-50 μM, preferably 0.5 μM-40 μM, more preferably 0.5 μM-30 μM, most preferably 0.5 μM-10 μM;
DZNeP: 1 nM-500 nM, preferably 5 nM-400 nM, more preferably 10 nM-300 nM, most preferably 10 nM-250 nM.
In some embodiments, the small molecule inhibitor of the STAT cell signaling pathway is a small molecule inhibitor targeting Src. In some embodiments, the small molecule inhibitor targeting Src is one or more selected from the group consisting of: Dasatinib, Quercetin, UM-164, KX2-391, and KX1-004. In some embodiments, the small molecule inhibitor targeting Src is one or more selected from the group consisting of: Dasatinib, UM-164, and KX1-004. In the applicant's research results, the above small molecule inhibitors of cell signaling pathways can well maintain the sternness of HSCs during in vitro expansion and the proportion of CD34+ cells, and the effect of the combinations of these small molecule inhibitors is superior to the small molecule inhibitor combinations reported in the prior art in the self-renewal and sternness maintenance of HSCs.
In this application, the applicant has found that a Src inhibitor can well maintain the sternness of HSCs in the in vitro expansion and culture of HSCs; and it was found that the effect of the combination of an inhibitor targeting HDAC and an inhibitor targeting Src is better than the effect of the combination of the small molecule inhibitors reported in the prior art and the effect of these small molecule inhibitors used alone in the in vitro expansion and culture of HSCs. The applicant's research results can realize the in vitro expansion of HSCs while maintaining a high proportion of sternness of HSCs, laying a foundation for the clinical application of HSCs.
The above-mentioned “sternness” of hematopoietic stem cells is an abbreviation for the characteristics of hematopoietic stem cells. Hematopoietic stem cells (HSCs) exhibit two major cell biological characteristics: self-renewal capacity and pluripotency. These properties of hematopoietic stem cells are referred to as “sternness” for short. To some extent, molecular phenotypes expressed on the cell surface of hematopoietic stem cells can reflect whether they maintain “sternness”. For example, if the phenotype of hematopoietic stem cells is CD34+CD45+CD90+CD45RA-CD38−, it means that they are LT-HSCs and maintain “sternness”. In this application, hematopoietic stem cells with CD34+CD45+CD90+CD45RA-CD38− phenotype are defined as LT-HSCs; if hematopoietic stem cells have CD34+CD45+CD90+CD45RA-CD38− phenotype, they are defined as maintaining or retaining the properties of hematopoietic stem cells, i.e., “sternness”.
The total cells refer to all progeny cells after culturing the initial CD34+ cells.
In the present invention, hematopoietic stem cells are contacted in vitro with a small molecule inhibitor of STAT cell signal transduction pathway, such as a small molecule inhibitor targeting Src or the small molecule inhibitor of the other cell signaling pathway, such as a small molecule inhibitor targeting HDAC, a small molecule inhibitor targeting PKC, and a small molecule inhibitor targeting JNK, thereby well maintaining the sternness of HSCs during in vitro expansion and the proportion of CD34+ cells in all HSCs, and in self-renewal, sternness maintenance and other aspects of HSCs, the effect of combinations of these small molecule inhibitors is superior to the effect of the small molecule combinations reported in the prior art, and the transplantation efficiency of cells treated with the above-mentioned small molecule inhibitors is significantly higher than the transplantation efficiency of cells treated with small molecule inhibitors reported in the prior art.
In
Preparation of reagents: H-lyse Buffer (1×) solution, and Wash Buffer (1×) solution. 5 ml of H-lyse Buffer (10×) stock solution (R&D, CAT. NO. WL1000) is taken, then adding 45 ml of deionized water (Edigene, filtrated with 0.22 μm filter membrane) to mix well to prepare an H-lyse Buffer (1×) solution. 5 ml of Wash Buffer (10×) stock solution (R&D, CAT. NO. WL1000) is taken, then adding 45 ml of deionized water to mix well to prepare a Wash Buffer (1×) solution.
Physiological saline is added to 10 ml of cord blood (Edigene) to a final volume of 30 ml. Human lymphocyte separation solution (Dakewe, CAT. NO. DKW-KLSH-0100) is added to the diluted blood, then centrifuging at 400 g for 30 min (setting acceleration speed 3, deceleration speed 0), sucking the buffy coat to centrifuge at 500 g for 10 min. The cell pellets are collected into a 50 ml centrifuge tube, adding 10 ml of H-lyse Buffer (1×) to lyse the red blood cells at room temperature for 10 min. Then 10 ml of Wash Buffer (1×) is added to stop the lysis reaction, adding physiological saline to make a final volume of 50 ml. The above 50 ml centrifuge tube is transferred to a high-speed centrifuge to centrifuge at 500 g for 10 min, discarding the supernatant, resuspending the cells with 50 ml of physiological saline (1% HSA) to mix well, and taking 20 μL of the cell suspension to a cell counter (Nexcelom, model: Cellometer K2), then transferring this centrifuge tube to high-speed centrifuge to centrifuge at 500 g for 10 min; discarding the supernatant, and adding the corresponding volume of magnetic beads (100 ul FcR/1*10{circumflex over ( )}8 cells and 100 ul CD34 MicroBeads/1*10{circumflex over ( )}8cells) according to the counting result. The specific operations are as follows: firstly, FCR blocking reagent (Miltenyi biotec, Cat. No. 130-100-453, the amount of the reagent is determined according to the result of cell counting) is added to resuspend the cells, then adding premixed CD34 MicroBeads (CD34 MicroBead Kit UltraPure, human: MiltenyiBiotec, Cat. No. 130-100-453) to mix well to incubate in a refrigerator at 4° C. for 30 min. Physiological saline (1% HSA) is added to the centrifuge tube to a final volume of 50 ml, transferring to a high-speed centrifuge to centrifuge at 500 g for 10 min. A magnetic separator (MiltenyiBiotec, model: 130-042-102) and a magnetic stand (MiltenyiBiotec, model: 130-042-303) are provided, adjusting the magnetic separator to a suitable height, and putting it into the MS Column (MiltenyiBiotec, Cat. No. 130-042-201) or LS column (MiltenyiBiotec, Cat. No. 130-042-401) (the type of the Column should be selected according to the number of cells, please refer to the relevant product instructions for details), then placing a 15 ml centrifuge tube (Corning, Cat. No. 430791) below to collect non-target cell suspension, rinsing the MS Column or LS Column with 1 ml (MS Column) or 3 ml (LS Column) of physiological saline (1% HSA). After centrifugation in the centrifuge tube in the above-mentioned high-speed centrifuge (Thermo, model: ST40), the supernatant is discarded, and the cells are resuspended in 1 ml (MS Column) or 3 ml (LS Column) of physiological saline (1% HSA), adding the cell suspension to each separation column (the amount of the separation column is determined according to the number of cord blood parts and the amount of cells); the centrifuge tube is washed with 1 ml (MS Column) or 3 ml (LS Column) of physiological saline (1% HSA), and the washing solution is added to the column.
The MS Column or LS Column is washed with 1 ml (MS Column) or 3 ml (LS Column) of physiological saline (1% HSA); repeating 3 times. The sorting column is transferred to the top of a new 15 ml centrifuge tube, adding 2 ml (MS Column) or 3 ml (LS Column) of physiological saline (1% HSA) to elute the target cells, and then adding 1 ml (MS Column) or 2 ml (LS Column) of physiological saline (1% HSA) to elute the target cells once again. 20 μL of cell suspension is taken to count in a cell counter (Nexcelom, model: Cellometer K2), and the remaining cell suspension is centrifuged at 400 g for 5 min, incompletely discarding the supernatant to keep 1 ml of the supernatant, and resuspending the cells. A new MS Column is taken to add 1 ml of physiological saline (1% HSA) to rinse it, transferring the cell suspension of the resuspended cells to the MS Column, repeating the above washing and elution steps to obtain 3 ml of the target cell suspension. 20 μL of cell suspension is taken to count in a cell counter (Nexcelom, model: Cellometer K2), calculating the total number of cells according to cell density and cell suspension volume; the remaining cell suspension is centrifuged at 400 g for 5 min, discarding the supernatant for later use.
A small molecule inhibitor stock solution is prepared according to the solubility and required solvent indicated in the instructions of the small molecule inhibitor product (see Table 1 for the Cat. Number of the small molecule inhibitor). Then the basal medium is prepared: SFEMII medium (stem cell, Cat. No. 09655)+50 ng/ml growth factor Flt-3L (PeProtech, Cat. No. 300-100UG)+50 ng/ml growth factor SCF (PeProtech, Cat. No. 300-07-100UG)+50 ng/ml growth factor TPO (PeProtech, Cat. No. 300-18-100UG)+10 ng/ml growth factor IL-6 (PeProtech, Cat. No. 200-06-20UG)+1% double antibody (HyClone, Cat. No. sv30010). Cultural media containing different concentrations of a small molecule inhibitor are prepared according to the preset concentration gradient of the small molecule inhibitor by using the stock solution and the basal medium.
Firstly, the prepared medium is added to a 24-well plate (Corning, Cat. No. 3473), 950 μl per well, placing it in a carbon dioxide incubator (Thermo, Model: 3111) to preheat; the spare HSCs prepared in Example 1 are resuspended with SFEMII+50 ng/ml Flt-3L+50 ng/ml SCF+50 ng/ml TPO+10 ng/ml IL-6+1% double antibody, the volume of medium to be added is calculated according to 50 μl cell suspension per well and 2*10{circumflex over ( )}5/ml cell density per well. For example, the final volume of the cell culture medium per well is 1 ml, the total number of cells per well is 2*10{circumflex over ( )}5 cells calculated according to the cell density per well, and the density of 50 μl of cell suspension added to each well is 4*10{circumflex over ( )}6/ml, adjusting the density of the spare HSCs prepared in Example 1 to the calculated density of the cell suspension for addition; the preheated medium is taken out from the incubator, adding 50 μl of the cell suspension to each well, and after mixing well, observing the cell state under the microscope (OLYMPUS, model: CKX53), and then putting it into an incubator for culture.
The antibodies used in this example and their sources are shown in Table 2.
20 μl of cells cultured for 6-7 days (D6-D7) in the above Example 2 is taken for counting, and a suspension of 2*10{circumflex over ( )}5 cells is taken to add into a 1.5 ml centrifuge tube according to the counting result; centrifuging at 400 g for 5 min, and discarding the supernatant. 100 μl of PBS (phosphate buffered saline, HyClone, Cat. No. SH30256.01) containing 1% HSA (human serum albumin, Guangdong Shuanglin, Cat. No. S10970069) is taken to resuspend the cells, vortexing to mix well for later use. Then, a control cell sample is collected, the number of cells and the collection method are the same as those of the sample cells to be tested. The control cells are set as the NC group and the ISO group respectively, and the cells are selected from any sample or mixed cells of the samples to be tested in this batch of experiments, depending on the number of cells. In the same batch of experiments, each control group does not have repeated detection. See Table 3 for group settings.
According to Table 3 above, antibodies are correspondingly added according to groups into the cell suspensions of the above-mentioned cell samples to be tested and control cell samples vortexing to mix well and incubate at room temperature for 15 min in the dark. After the 15 min incubation, 1 ml of PBS containing 1% HSA is added to each experimental sample to mix well, centrifuging at 400 g for 5 min at room temperature. After centrifugation, the supernatant is discarded, and the cells are resuspended in 100 μl of PBS containing 1% HSA for each experimental sample, storing the samples at room temperature away from light before testing. Flow cytometry is used to detect them.
The test results are analyzed as follows: 1) the target cell population is CD34+CD45+CD45RA-CD90+CD38− cell population; 2) the determination of the logic gate and gate position is shown in
On the umbilical cord blood-derived CD34+ cells sorted in Example 1, the optimal concentrations of small molecule inhibitors and the ability to maintain the stemness of HSCs are screened according to the same method as in Example 2. After 6-7 days of small molecule induction, the expression of cell surface markers (CD34+CD45+CD90+CD45RA-CD38−) of long-term hematopoietic stem cells (LT-HSCs) is detected by flow cytometry according to the same method as in Example 3.
A total of five rounds of screening are carried out in this example, and the inhibitors and test concentrations for each round of screening are shown in Table 4, Table 5, Table 6, Table 7, and Table 8, and the results are shown in
The results in
The results in
The results in
The results in
The results in
To sum up: in this Example, a total of 5 small molecules that can maintain the sternness of LT-HSCs and a high proportion of CD34+ cells are screened out; and they are respectively VPA and SAHA which target HDAC, Dasatinib targeting Src., Enzastaurin targeting PKC, and JNK-IN-8 targeting JNK.
On the umbilical cord blood-derived CD34+ cells sorted in Example 1, the screening of the best combination of two small molecule inhibitors to maintain the sternness of HSCs is carried out according to the same method as in Example 2. After 6-7 days of induction with a combination of small molecule inhibitors, the expression of LT-HSCs cell surface markers (CD34+CD45+CD90+CD45RA-CD38−) is detected by flow cytometry according to the same method as in Example 3.
The small molecule SAHA that can significantly maintain the sternness of HSCs screened according to the above Example 4 is respectively combined with other inhibitors. The specific combination and its concentration are shown in the relevant drawings, and the results are shown in
The results in
The results in
The results in
The results in
The results in
In summary, in the screening of the combination of two small molecule inhibitors, the combinations that can maintain the sternness of LT-HSCs and the proportion of CD34+ cells are screened out as follows: SAHA+Dasatinib, SAHA+DZNeP, SAHA+EPZ004777, SAHA+JNK-IN-8, and SAHA+VPA.
On the umbilical cord blood-derived CD34+ cells sorted in Example 1, the screening of the best combination of three small molecule inhibitors to maintain the sternness of HSCs is performed according to the same method as in Example 2. After 6-7 days of induction with a combination of small molecule inhibitors, the expression of LT-HSCs cell surface markers (CD34+CD45+CD90+CD45RA-CD38−) is detected by flow cytometry according to the same method as in Example 3.
(1) The combinations of SAHA+EPZ004777 and SAHA+DZNeP screened in Example 5 (which can maintain the sternness of HSCs) are subjected to the combination of three small molecule inhibitors, and the results are shown in
The results in
(2) The combinations of SAHA+EPZ004777, SAHA+DZNeP, SAHA+JNK-IN-8 and SAHA+Dasatinib screened in Example 5 (which can maintain the sternness of HSCs) are subjected to the combination of three small molecule inhibitors, and the results are shown in
The results in
The results in
To sum up, in terms of maintaining the sternness of LT-HSCs, most of the combinations of three small molecule inhibitors are not as effective as the combination of two small molecule inhibitors, i.e., SAHA+Dasatinib; while among the combinations of three small molecule inhibitors, the best combinations are SAHA+EPZ004777+DZNeP, and SAHA+Dasa+VPA.
On the umbilical cord blood-derived CD34+ cells sorted in Example 1, according to the same method as Example 2, comparison between an inhibitor used alone and inhibitors used in combination for the screened inhibitors SAHA, VPA, and Dasatinib and the inhibitors UM171 and SR1 reported in the literatures (Fares I, et al. Science. 2014; Boitano A E, et al. Science. 2010;) is performed. After 6-7 days of induction with small molecule inhibitor(s), the expression of LT-HSCs cell surface markers (CD34+CD45+CD90+CD45RA-CD38−) is detected by flow cytometry according to the same method as in Example 3; and the results are shown in
The results in
To sum up, in terms of maintaining the sternness of LT-HSCs and the proportion of CD34+ cells, the combination of two small molecule inhibitors, i.e., SAHA+Dasatinib or VPA+Dasatinib, is more effective than the small molecule inhibitor used alone, the combination of two small molecule inhibitors, i.e., SAHA+SR1 or SAHA+UM171, and the combination of three small molecule inhibitors, i.e., SAHA+DZNeP+EPZ004777.
In this example, qualitative and quantitative detections are performed by using colony-forming unit (CFU) to detect the in vitro function of cord blood-derived hematopoietic stem cells after induction with a small molecule inhibitor, thereby verifying the in vitro differentiation potential.
Firstly, 100 mL of MethoCult™ H4034 Optimum (stem cell, Cat. No. 04034) is aliquoted, then thawing at 2-8° C. overnight; shaking vigorously for 1-2 min and standing for 10 min until the bubbles rise to the liquid level. After tightly fitting the 50 mL syringe needle to the 5 mL disposable syringe, the medium is aspirated to 1 mL, pushing out the syringe completely to exhaust the gas in the syringe, and re-absorbing 3 mL into each 15 mL centrifuge tube (Corning, Cat. No. 430791); storing at 2-8° C. for 1 month and at −20° C. for a long time. Do not freeze and thaw repeatedly.
3 mL of medium MethoCult™ H4034 Optimum is prepared then thawing overnight at room temperature (15-25° C.) or 2-8° C.
Cell seeding is performed. The cell suspension undergoing 7 days of expansion and culture after induction with small molecule inhibitor(s) (cord blood-derived CD34+ hematopoietic stem cells after induction with small molecule inhibitor(s)) is taken for cell counting, then aspirating the cell suspension at 100 times the seeding density according to the counting results (for example, the seeding density is 100 cells/well/3 ml, and 10000 cells should be collected), adding to 1 ml of 2% FBS (Gibco, Cat. No. 16000-044)-IMDM (Gibco, Cat. No. 12440-053) medium to mix well for use. After mixing the above cells, 50 pl of the cell suspension is aspirated into 0.5 mL of IMDM (2% FBS) to resuspend the cells (equivalent to 10-fold dilution of the cell suspension). After mixing well, 100 μl of the cell suspension (100 cells) is taken to add into 3 mL of MethoCult™ H4034 Optimum; vortexing for at least 4s and standing for 10 min until the bubbles rise to the liquid level. 3 cc Syringes (Stem cell, Cat. No. 28240) is used in conjunction with Blunt-End Needles 16 Gauge (Stemcell, Cat. No. 28110) to aspirate the obtained cell suspension to 1 mL, then pushing it out of the syringe to exhaust the gas in the syringe, and re-absorbing all the obtained cells suspension to inject 3 mL into one well of SmsrtDishTM-6 (stem cell, Cat. No. 27370, 6-well plate), and slowly tilting the 6-well plate so that the cell suspension evenly covers the bottom of the well. After inoculating all cells as described above, 3 ml of sterile PBS is added to gaps between wells of the 6-well plate to prevent the medium from drying up. The 6-well plate is covered with a lid, then placing the plate in a carbon dioxide incubator (Thermo, model: 3111) to culture for 14 days at 37° C., 5% CO2, and 95% relative humidity.
Colonies are observed on day 7 and day 14 of the culture, and colonies are counted with a STEMgridTM-6 counting grid (stem cell, Cat. No. 27000) after culturing for 14 days. The criteria for determining colonies are as follows (different types of colonies can reflect the ability of HSCs to form colonies and maintain the stemness):
CFU-GEMM (CFU-G, CFU-E, CFU-MM): granulocyte-erythrocyte-macrophage-megakaryocyte colony forming unit. A colony contains erythrocytes and 20 or more non-erythrocytes (granulocytes, macrophages, and/or megakaryocytes), usually the erythrocytes are located in the center of the colony and surrounded by non-erythrocytes, and non-erythrocytes can also be concentrated on one side of the erythrocytes. Generally, colonies of CFU-GEMM are larger than those of CFU-GM or BFU-E and they rare in most cell samples (usually 10% of the total colonies).
CFU-GM: a colony contains more than 20 granulocytes (CFU-G) and/or macrophages (CFU-M). Without appearing red or brown, individual cells within a colony are often distinguishable, especially at the edge of the colony, and a large colony may have one or more dense dark nuclei. Erythropoietin (EPO) is not required for the growth and differentiation of this colony.
BFU-E: burst erythrocyte colony-forming unit, forming colonies consist of single or multiple cell clusters, each colony containing >200 mature erythrocytes. When cells are hemoglobinated they appear red or brown, making it difficult to distinguish individual cells within each cluster, BFU-E are more immature progenitor cells whose growth require erythropoietin (EPO) and other cytokines, especially interleukin 3 (IL-3) and stem cell factor (SCF) for optimal growth of their colonies.
CFU-E: erythrocyte colony-forming unit, which can form 1-2 cell clusters containing 8-200 red blood cells, when the cells are hemoglobinized they appear red or brown, making it difficult to distinguish individual cells within the colony. CFU-E are progenitors of the mature erythroid lineage, and they require erythropoietin (EPO) to promote their differentiation.
The Comparison of in vitro clonogenic ability between an Inhibitor Used Alone and Inhibitors Used in Combination for the screened inhibitors SAHA, VPA, and Dasatinib and the inhibitors UM171 and SR1 reported in the literatures is carried out on the umbilical cord blood-derived CD34+ cells sorted in Example 1. In vitro clone (CFU) formation is detected by the same method as in Example 8 after inducing the cells with small molecule inhibitors for 7 days, and the number of clones is counted 14 days after inoculation of the cells, and the CFU-GEMM is analyzed. The results are shown in
The results in
In conclusion, in terms of in vitro clonogenic ability, the combinations of VPA+Dasatinib and SAHA+Dasatinib are superior to the small molecule SR1, UM171 reported in the known literatures when an inhibitor is used alone or used in combination with SAHA.
On the umbilical cord blood-derived CD34+ cells sorted in Example 1, other small molecule inhibitors targeting Src are screened according to the same method as in Example 2. After 6-7 days of induction with a combination of small molecule inhibitors, the expression of LT-HSCs cell surface markers (CD34+CD45+CD90+CD45RA-CD38−) is detected by flow cytometry according to the same method as in Example 3. The results are shown in
The results in
On the umbilical cord blood-derived CD34+ cells sorted in Example 1, a comparison of in vitro expansion and the ability to maintain the sternness of hematopoietic stem cells for the screened inhibitor Dasatinib and the inhibitors UM171 and SR1 reported in the literatures is carried out. After 6-8 days of induction with the small molecule inhibitor, the expression of LT-HSCs cell surface markers (CD34+CD45+CD90+CD45RA-CD38−) is detected by flow cytometry according to the same method as in Example 3. 20 μL of cell suspension is respectively taken on day 2, day 4, day 6 and day 8 of the culture to count in a cell counter (Nexcelom, model: Cellometer K2), calculating the final absolute number of CD34+ cells and LT cells on day 8 (absolute number of cells=cell proportion*total number of cells), the results are shown in
The results in
On the umbilical cord blood-derived CD34+ cells sorted in Example 1, a comparison of the in vivo hematopoietic system reconstitution ability between the screened small molecule inhibitor Dasatinib and the inhibitor SR1 reported in the literature which are used alone is performed. The concentrations and groups of small molecule inhibitors used in this Example are shown in Table 10.
Preparation of cell culture medium: as for SFEMII medium+50 ng/ml growth factor Flt-3L+50 ng/ml growth factor SCF+50 ng/ml growth factor TPO+10 ng/ml growth factor IL-6+1% double antibody, catalog numbers of the used medium, growth factors, and double antibodies, etc. are the same as those described in Example 2. Different small molecule inhibitors are respectively added according to the groups set in Table 10.
The prepared cell culture medium is added to a 24-well plate, 950 μl per well, then placing it in a carbon dioxide incubator to preheat; the prepared HSCs in Example 1 are resuspended with SFEMII+50 ng/ml Flt-3L+50 ng/ml SCF+50 ng/ml TPO+10 ng/ml IL-6+1% double antibody, then calculating the volume of the added medium according to 50 ul of cell suspension per well, and the cell density per well of 1*10{circumflex over ( )}5/ml; taking out the preheated medium from the incubator, adding 50 μl of cell suspension to each well to mix well, and observing the cell state under a microscope, and then putting it into an incubator for culture. The amount of initial cultured cells for transplantation in each mouse is 1*10{circumflex over ( )}5/mouse, and the cells expanded in each well of the 24-well plate is sufficient for transplantation into one mouse. During the cell culture process, cells are counted every other day, the counting method and cell counter are the same as in Example 1, making sure that the cell density does not exceed 8*10{circumflex over ( )}5/ml. If the cells are too dense, the cells in a well should be divided in time and fresh medium is added.
The expression of LT-HSCs cell surface markers (CD34+CD45+CD90+CD45RA-CD38−) is detected according to the same method as in Example 3 after treating the cells with the small molecule inhibitor for 7 days.
Mice are prepared, with 8 mice per group. Mice are purchased from Beijing Weitongda Biotechnology Co., Ltd., the strain is NPG (NOD-Prkdcscidll2rgnull/Vst), 6-week-old female mice, and the weight difference between the mice is controlled within 3 g. The mice are irradiated with a half-lethal dose before cell transplantation, and the irradiation dose is 1.6 Gy.
The cultured cell suspension (the initial culture cell amount is 1*10{circumflex over ( )}5/ml) are collected to centrifuge at 400 g for 5 min, discarding the supernatant, then resuspending the cells with 100 μl of physiological saline (1% HSA), and injecting the cells into an irradiated NPG mice through the tail vein, different groups of mice are labeled.
After the cells are transplanted into the mice, the peripheral blood of the mice is respectively collected at week 4, week 8, and week 12, and the proportion of human CD45 is detected by flow cytometry; the mice are sacrificed at week 16, and the peripheral blood, bone marrow cells and spleen cells of the mice are collected to detect the proportion of human CD45, human CD19, human CD3, human CD33 and human CD56 by flow cytometry. Antibodies and 7-AAD dye used in this example and their sources are shown in Table 11.
The proportion of human CD45 in peripheral blood of mice is detected by flow cytometry, and the set cell detection groups are shown in Table 12.
Preparation of 1× erythrocyte lysate: 5 ml of RBC Lysis/Fixation Solution 10× stock solution (Biolegend, Cat. No. 422401) is taken, adding 45 ml of deionized water (Edigene, iltered with a 0.22 μm filter) to mix well to prepare 1× erythrocyte lysate.
The peripheral blood of mice (about 100 μl) is collected, and antibodies are added according to the groups set in Table 12, vortexing to mix well and incubating at room temperature for 15 min in the dark. After the incubation is completed, 1.2 ml of 1× red blood cell lysis solution is respectively added to the NC and each sample, vortexing to mix well, and lysing at room temperature for 15 min in the dark, during this period the sample centrifuge tube is inverted every 3 min. After the lysis is completed, the sample solutions are centrifuged at 400 g for 5 min at room temperature. After the centrifugation, the supernatant is discarded, adding 1 ml of PBS containing 1% HSA to each experimental sample to mix well, centrifuging at 400 g for 5 min at room temperature. After centrifugation, the supernatant is discarded, adding 100 μl of PBS containing 1% HSA and 5 μl of 7-AAD dye to each experimental sample to mix well by vortexing, and incubating at room temperature for 5 min in the dark. After the incubation, 1 ml of PBS containing 1% HSA is added to the NC and each sample to mix well, centrifuging at 400 g for 5 min at room temperature. After the centrifugation, the supernatant is discarded, then adding 100 ul of PBS containing 1% HSA to each experimental sample to resuspend the cells; the samples are stored at room temperature in the dark before testing, and detected by flow cytometry.
The test results are analyzed as follows: 1) the target cell population is human CD45+ cell population; 2) the determination of the logic gate and gate position is shown in
The proportions of human CD45, human CD19, human CD3, human CD33 and human CD56 in peripheral blood, bone marrow cells and spleen cells of mice are detected by flow cytometry. See Table 13 for the set cell detection groups.
The peripheral blood of mice (about 100 μl) is collected, and antibodies are respectively added according to the groups set in Table 13. Subsequent blood sample processing is consistent with the above-mentioned operation for detecting the proportion of human CD45 in peripheral blood of mice. After the operation, a flow cytometer is used for detection.
The mice are sacrificed by cervical dislocation, and the tibia and femur of one hind leg of the mice are taken. Ophthalmic scissors and ophthalmic forceps are used to cut off both ends of the tibia and femur respectively to expose the marrow cavity. 1 ml syringe is used to draw the pre-cooled PBS containing 1% HSA, the needle is injected into one end of the bone marrow cavity, and the PBS is injected forcefully to flush out the bone marrow cells from the other end of the bone marrow cavity. Tibial and femoral marrow cavities are respectively rinsed with 2 ml of PBS. The cell suspension of bone marrow is repeatedly blown and sucked with a pipette, then filtering through a 40 um cell mesh (BD, catalog number: 352340), centrifuging at 400 g for 5 min at room temperature. After centrifugation, the supernatant is discarded, and the bone marrow cells are used for later use.
The mice are sacrificed by cervical dislocation, and the mouse spleen is removed and placed in pre-cooled PBS containing 1% HSA. The spleen is cut with ophthalmic scissors, and the spleen tissue suspension is repeatedly blown and sucked with a pipette, then filtering through a 40 μm cell mesh, and centrifuging at 400 g for 5 min at room temperature. After centrifugation, the supernatant is discarded, and the spleen cells are used for later use.
1 ml of 1× erythrocyte lysate is added to the spare bone marrow cells and spleen cells, vortexing to mix well, and lysing at room temperature for 15 min, during this period the sample centrifuge tube is inverted every 3 min. After the lysis, 4 ml of PBS containing 1% HSA is added to each sample to centrifuge at room temperature for 5 min at 400 g. After centrifugation, the supernatant is discarded, a and 1 ml of PBS containing 1% HSA is added to each sample to mix well by vortexing. 100 pl of cell suspension is taken out from each sample, then respectively adding antibodies according to the groups in Table 13, vortexing to mix well, and incubating at room temperature for 15 min in the dark. After the incubation, 5 μl of 7-AAD dye is added to each experimental sample, vortexing to mix well, and incubating at room temperature for 5 min in the dark. After the incubation, 1 ml of PBS containing 1% HSA is added to the NC and each sample to mix well, then centrifuging at 400 g for 5 min at room temperature. After centrifugation, the supernatant is discarded, and 100 pl of PBS containing 1% HSA is added to each experimental sample to resuspend the cells. The samples are stored at room temperature in the dark before testing, and detected by flow cytometry.
The test results are analyzed as follows: 1) the target cell population is human CD45+ cell population, human CD19+ cell population, human CD3+ cell population, human CD33+ cell population, and human CD56+ cell population; 2) the determination of the logic gate and gate position is shown in the
The results in
The results in
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/125687 | Dec 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/136790 | 12/16/2020 | WO |
