SMALL MOLECULE COMPOUNDS FOR AMPLIFYING HEMATOPOIETIC STEM CELLS, AND COMBINATION THEREOF
20230027247 · 2023-01-26
Assignee
Inventors
- Riguo FANG (Beijing, CN)
- Huihui YANG (Beijing, CN)
- Zhongyu SHI (Beijing, CN)
- Pengfei YUAN (Beijing, CN)
- Lingling YU (Beijing, CN)
Cpc classification
C12N2501/125
CHEMISTRY; METALLURGY
A61K31/167
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
C12N5/0647
CHEMISTRY; METALLURGY
A61K31/167
HUMAN NECESSITIES
A61K31/5377
HUMAN NECESSITIES
A61K31/352
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
International classification
Abstract
Provided are small molecule inhibitors for amplifying hematopoietic stem cells (HSCs) and a combination thereof. The small molecule inhibitors and the combination thereof can maintain the sternness of hematopoietic stem cells while promoting the in vitro amplification of hematopoietic stem cells (HSCs).
Claims
1. A method for promoting the proliferation of hematopoietic stem cells (HSCs) and/or maintaining the stemness of the HSCs, comprising in vitro contacting the HSCs with a culture medium comprising a small molecule inhibitor of the signal transducer and activator of transcription (STAT) cell signaling pathway.
2. The method according to claim 1, wherein the small molecule inhibitor of the STAT cell signaling pathway is a small molecule inhibitor targeting Src.
3. The method according to claim 2, wherein 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.
4. The method according to claim 2, wherein the small molecule inhibitor targeting Src is used in combination with a small molecule inhibitor of the other cell signaling pathway.
5. The method according to claim 4, wherein the small molecule inhibitor of the other cell signaling pathway is one or more selected from the group consisting of: a small molecule inhibitor targeting histone deacetylase (HDAC), a small molecule inhibitor targeting protein kinase C (PKC), and a small molecule inhibitor targeting c-Jun N-terminal kinase (JNK).
6. (canceled)
7. The method according to claim 5, wherein the small molecule inhibitor targeting Src is used in combination with the small molecule inhibitor valproic acid (VPA) targeting HDAC, the small molecule inhibitor suberoylanilide hydroxamic acid (SAHA) targeting HDAC, the small molecule inhibitor Enzastaurin targeting PKC, or the small molecule inhibitor JNK-IN-8 targeting JNK.
8. The method according to claim 5, wherein the small molecule inhibitor targeting Src is Dasatinib.
9. The method according to claim 7, wherein the small molecule inhibitor targeting Src is Dasatinib, and wherein Dasatinib is used in combination with VPA or SAHA.
10. The method according to claim 2, wherein the small molecule inhibitor targeting Src is used alone or in combination with one or more other small molecule inhibitors to maintain the HSCs with a CD34+CD45+CD90+CD45RA-CD38− phenotype accounting for more than about 8% of all HSCs.
11. The method according to claim 2, wherein the small molecule inhibitor targeting Src is used alone or in combination with one or more other small molecule inhibitors to maintain CD34+ HSCs accounting for more than about 65% of all HSCs.
12. A composition for maintaining the stemness of HSCs, comprising a small molecule inhibitor of the STAT cell signaling pathway.
13-15. (canceled)
16. The composition according to claim 12, further comprising a small molecule inhibitor of the other cell signaling pathway selected from one or more of a small molecule inhibitor targeting HDAC, a small molecule inhibitor targeting PKC, and a small molecule inhibitor targeting JNK.
17. The composition according to claim 16, wherein 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.
18. The composition according to claim 12, further comprising Serum-Free Expansion Medium II (SFEM II), growth factor Fms Related Tyrosine Kinase 3 Ligand (Flt-3L), growth factor Stem Cell Factor (SCF), growth factor thrombopoietin (TPO), and/or growth factor interleukin 6 (IL-6).
19-24. (canceled)
25. A composition for maintaining the stemness of HSCs, comprising any combination selected from: SAHA+EPZ004777, SAHA+3-Deazaneplanocin A (DZNeP), SAHA+Dasatinib, VPA+Dasatinib, SAHA+JNK-IN-8, SAHA+VPA, SAHA+EPZ004777+DZNeP, or SAHA+VPA+Dasatinib.
26. The composition according to claim 25, wherein the composition maintains the HSCs with CD34+CD45+CD90+CD45RA-CD38− phenotype accounting for more than about 8% of all HSCs.
27. The composition according to claim 25, wherein the composition maintains CD34+HSCs accounting for more than about 65% of all HSCs.
28. The composition according to claim 25, further comprising SFEM II, growth factor Flt-3L, growth factor SCF, growth factor TPO, and/or growth factor IL-6.
29. The composition according to claim 12, comprising one or more of: Dasatinib in an amount of from about 0.1 μM to about 50 μM; SAHA in an amount of from about 10 nM to about 20 μM; VPA in an amount of from about 10 μM to about 2000 μM; JNK-IN-8 in an amount of from about 0.1 μM to about 20 μM; EPZ004777 in an amount of from about 0.1 μM to about 50 μM; DZNeP in an amount of from about 1 nM to about 500 nM; UM-164 in an amount of from about 0.1 μM to about 1000 μM; KX2-391 in an amount of from about 0.1 nM to about 1000 nM; and KX1-004 in an amount of from about 0.1 μM to about 1000 μM.
30. The composition according to claim 25, wherein the concentration of each component in the composition is: Dasatinib in an amount of from about 0.1 μM to about 50 μM; SAHA in an amount of from about 10 nM to about 20 μM; VPA in an amount of from about 10 μM to about 2000 μM; JNK-IN-8 in an amount of from about 0.1 μM to about 20 μM; EPZ004777 in an amount of from about 0.1 μM to about 50 μM; or DZNeP in an amount of from about 1 nM to about 500 nM.
31. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
Example 1: Sorting CD34+HSCs from Umbilical Cord Blood for Subsequent Small Molecule Inhibitor Screening
[0069] 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.
[0070] 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.
[0071] 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.
Example 2: Concentration Testing and Screening of Small Molecule Inhibitor
[0072] 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.
[0073] 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.
TABLE-US-00001 TABLE 1 Small Molecule Inhibitors Cat. No. (The following are from Selleck) Name of small molecule inhibitor Action pathway/target S1021 Dasatinib (Dasa) Src, c-kit, Abl S2391 Quercetin Sirtuin, Src, PKC, PI3K S8706 UM-164 Src, P38α/β S2700 KX2-391 Src S6500 KX1-004 Src S1047 SAHA HDAC S1999 Sodium butyrate HDAC S3944 Valproic acid (VPA) HDAC S7595 Santacruzamate A (SIS3 HCl) HDAC S7085 IWP-2 Porcn in the Wnt pathway S7086 IWR-1-endo Wnt pathway S8007 VE821 ATR S7050 AZ-20 ATR S7079 SGC 0946 DOTIL methyltransferase S7353 EPZ004777 DOTIL methyltransferase S7307 GSK2606414 EIF2AK3 (PERK, protein kinase R-like endoplasmic reticulum kinase) S7400 ISRIB (trans-isomer) PERK S2924 CHIR-99021 (CT99021) HCl GSK3a/(glycogen synthetase kinase) S2621 AZD5438 CDK1/2/9 (cyclin-dependent kinases1/2/9) S4901 JNK-IN-8 JNK pathway S7508 JNK Inhibitor IX JNK pathway s7483 DMOG HIF prolyl hydroxylase S1623 Acetylcysteine ROS (reactive oxygen species), TNFa (tumor necrosis factor) S3114 Vitamin C ROS s5433 Sodium succinate ROS s2284 Colchicine microtubule polymerization s2775 Nocodazole microtubule polymerization, Abi S4505 Vinblastine sulfate microtubule formation, nAChR (subunit nicotinic acetylcholine receptor) S3633 Pyrrolidinedithiocarbamate NF-kB ammonium S4073 Sodium 4-Aminosalicylate(S-4-A) NF-kB S1067 SB431542 TGFPRI/ALK5 S1459 Thiazovivin ROCK (Rho-associated kinase) S1049 Y27632 ROCK (Rho-associated kinase) S1076 SB203580 P38 MAPK (mitogen-activated protein kinase) S2449 Forskolin cAMP Activator S7858 Dibutyryl-cAMP (Bucladesine) PKA (cAMP dependent protein kinase A) sl 907 Metronidazole DNA synthesis SI 992 Fluticasone propionate(FLU) Glucocorticoid receptor s2250 (-)-EpigallocatechinGallate telomerase, DNA methyltransferase s2923 Salubrinal eIF2a S3925 (-)-Epicatechingallate VEGFR2 (vascular endothelial growth factor 2) S4991 Valpromide VPA derivative, target unknown S7120 3-deazaneplanocin A (DZNeP) HCl S-adenosylhomocysteinehydrolase,Histone methyltransferase S7315 PFI-3 PB 1(5) bromodomains, SMARCA4/2A/2B S7608 UM171 unknown S8287 CPI-455 HCI KDM5 (histone demethylase) s8615 Sodium dichloroacetate (DCA) PDK4/2 (pyruvate dehydrogenase kinase) S5742 Deferoxamine mesylate unknown S2858 SRI AHR (aryl hydrocarbon receptor) S4757 Dihydrotestosterone(DHT) Androgen receptor S8280 IMR-1 Notch pathway S2915 GW9662 PPARy (peroxisome proliferator- activated receptor) S3003 PGE2 Wnt pathway S1055 Enzastaurin PKC S2911 Go 6983 PKC (protein kinase C) S2776 CPI-613 PDH (pyruvate dehydrogenase), a-ketoglutarate dehydrogenase
Example 3: Flow Cytometry Detection of Stemness of HSCs and Maintenance of CD34+
[0074] The antibodies used in this example and their sources are shown in Table 2.
TABLE-US-00002 TABLE 2 Antibodies Antibody name Manufactor Cat. No. APC/Cy7 anti-human CD45 Biolegend 304014 APC anti-human CD38 Biolegend 356606 Brilliant Violet 510™ Biolegend 343528 anti-human CD34 PE anti-human CD90 (Thy1) Biolegend 328110 FITC anti-human CD45RA Biolegend 304106 APC Mouse IgG2a, κ Isotype Ctrl Biolegend 400220 APC/Cyanine7 Mouse IgGl, κ Isotype Ctrl Biolegend 400128 PE Mouse IgG2a, κ Isotype Ctrl Biolegend 400212 FITC Mouse IgG2b, κ Isotype Ctrl Biolegend 402208 Brilliant Violet 510™ Biolegend 400268 Mouse IgG2a, κ Isotype Ctrl
[0075] 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.
TABLE-US-00003 TABLE 3 Group settings Group Number of cells Name of the added antibody Antibody quantity NC 2 × 10{circumflex over ( )}5 — — ISO 2 × 10{circumflex over ( )}5 APC Mouse IgG2a, k Isotype Ctrl 2 μl APC/Cyanine7 Mouse IgGl, k Isotype Ctrl 2 μl PE Mouse IgG2a, k Isotype Ctrl 2 μl FITC Mouse IgG2b, k Isotype Ctrl 2 μl Brilliant Violet 510™ Mouse IgG2a, k Isotype Ctrl 2 μl FMO38 2 × 10{circumflex over ( )}5 APC/Cy7 anti-human CD45 2 μl Brilliant Violet 510™ anti-human CD34 2 μl PE anti-human CD90 (Thyl) 2 μl FITC anti-human CD45RA 2 μl FMO90 2 × 10{circumflex over ( )}5 APC/Cy7 anti-human CD45 2 μl APC anti-human CD38 2 μl Brilliant Violet 510™ anti-human CD34 2 μl FITC anti-human CD45RA 2 μl sample 2 × 10{circumflex over ( )}5 APC/Cy7 anti-human CD45 2 μl APC anti-human CD38 2 μl Brilliant Violet 510™ anti-human CD34 2 μl PE anti-human CD90 (Thyl) 2 μl FITC anti-human CD45RA 2 μl
[0076] 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.
[0077] 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
Example 4: Single Molecule Screening
[0078] 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.
[0079] 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
[0080] The results in
[0081] The results in
[0082] The results in
[0083] The results in
[0084] The results in
[0085] 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.
TABLE-US-00004 TABLE 4 First Round of Screening Name of small molecule inhibitor Test concentration VE821 0.1 μM, 1 μM, 5 μM, 10 μM AZ20 0.2 μM, 1 μM, 5 μM, 10 μM PFI-3 0.2 μM, 2 μM, 5 μM, 10 μM Sodium 4-Aminosalicylate (S-4-A) 0.1 mM, 1 mM, 5 mM, 10 mM PDTC 1 nM, 5 nM, 10 nM, 50 nM SAHA 0.1 μM, 1 μM, 5 μM, 10 μM Santacruzamate A(SIS3 HCL) 0.1 μM, 1 μM, 5 μM, 10 μM SR1 0.1 μM, 1 μM, 5 μM, 10 μM
TABLE-US-00005 TABLE 5 Second Round of Screening Name of small molecule inhibitor Test concentration SAHA 1 μM SR1 5 μM UM171 350 nM PGE2 10 μM GW9662 1 μM FLU 1 μM
TABLE-US-00006 TABLE 6 Third Round of Screening Name of small molecule inhibitor Test concentration SAHA 1 μM Vitamin C 5 μg/ml, 25 μg/ml, 50 μg/ml EPZ004777 0.5 μM, 5 μM, 10 μM Forskolin 5 μM, 10 μM, 20 μM CPI-455 1 μM, 5 μM, 10 μM DZNeP 10 nM, 50 nM, 250 nM CHIR-99021 1 μM, 3 μM, 10 μM Butyrate 50 μM, 250 μM,500 μM, SB203580 1 μM, 5 μM, 10 μM IWP-2 1 μM, 5 μM, 10 μM IWR-l-endo 1 μM, 5 μM, 10 μM JNK-inhibitor iX 1 μM, 5 μM, 10 μM Dibutyryl-cAMP 1 μM, 5 μM, 10 μM Thiazovivin 1 μM, 5 μM, 10 μM IMR-1 1 μM, 5 μM, 10 μM SB431542 1 μM, 5 μM, 10 μM Quercetin 0.5 μM, 1 μM, 5 μM
TABLE-US-00007 TABLE 7 Fourth Round of Screening Name of small molecule inhibitor Test concentration SAHA 1 μM Dasatinib 0.5 μM 5 μM 10 μM SGC0496 1 μM 5 μM 10 μM JNK-IN-8 1 μM 2 μM 10 μM Enzastaurin(LY317615) 1 μM 5 μM 10 μM
TABLE-US-00008 TABLE 8 Fifth Round of Screening Name of small molecule inhibitor Test concentration SAHA 1 μM Valproic acid 100 μM 500 μM 1000 μM Valpromide 100 μM 500 μM 1000 μM AZD5438 1 μM 5 μM 10 μM Go 6983 1 μM 5 μM 10 μM Sodium succinate 5 μM 10 μM 50 μM Sodium dichloroacetate (DCA) 0.1 mM 1 mM 5 mM Salubrinal 1 μM 5 μM 10 μM CPI-613 5 μM 10 μM 50 μM ISRIB(trans-isomer) 1 μM 5 μM 10 μM Acetylcysteine 1 μM 5 μM 10 μM GSK2606414 10 nM 50 nM 100 nM DMOG 1 μM 5 μM 10 μM Metronidazole 1 μM 5 μM 10 μM DHT 1 nM 5 nM 50 nM Nocodazole 1 μM5 μM 10 μM Vinblastine sulfate 1 μM5 μM 10 μM Epigallocatechin Gallate 1 μM5 μM 10 μM Deferoxamine mesylate: 10 nM 50 nM 500 nM Epicatechin gallate 10 nM 50 nM 500 nM Colchicine 50 nM 500 nM 1000 nM Y27632 5 μM 10 μM 20 μM
Example 5: Screening of the Combination of Two Small Molecule Inhibitors
[0086] 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.
[0087] 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
[0088] The results in
[0089] The results in
[0090] The results in
[0091] The results in
[0092] The results in
[0093] 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.
Example 6: Screening of the Combination of Three Small Molecule Inhibitors
[0094] 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.
[0095] (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
[0096] The results in
[0097] (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
[0098] The results in
[0099] The results in
[0100] 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.
Example 7: 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
[0101] 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
[0102] The results in
[0103] 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.
Example 8: CD34+Hematopoietic Stem Cell Colony Formation and Culture
[0104] 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.
[0105] 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.
[0106] 3 mL of medium MethoCult™ H4034 Optimum is prepared then thawing overnight at room temperature (15-25° C.) or 2-8° C.
[0107] 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% CO.sub.2, and 95% relative humidity.
[0108] 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):
[0109] 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).
[0110] 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.
[0111] 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.
[0112] 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.
Example 9: 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
[0113] 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
[0114] The results in
[0115] 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.
Example 10: Verification of the Effect of Src Pathway Inhibitors
[0116] 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
TABLE-US-00009 TABLE 9 Src pathway inhibitors and selected concentrations Name of small the molecule inhibitor Tested concentration SAHA 1 μM UM-164 1 μM, 5 μM, 10 μM KX2-391 10 nM, 50 nM, 100 nM KX1-004 10 μM, 50 μM, 100 μM
[0117] The results in
Example 11: Comparison of In Vitro Expansion and the Ability to Maintain the Stemness of Hematopoietic Stem Cells for the Screened Inhibitor Dasatinib and the Inhibitors UM171 and SR1 Reported in the Literatures
[0118] 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
[0119] The results in
Example 12: Validation of the Effect on In Vivo Transplantation of Hematopoietic Stem Cells for the Screened Inhibitor Dasatinib and the Inhibitor SR1 Reported in the Literatures
[0120] 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.
TABLE-US-00010 TABLE 10 Small molecule inhibitor concentrations Group Concentration of small molecule inhibitor Mock — SRI 5 μM Dasatinib 50 nM
[0121] 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.
[0122] 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.
[0123] 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.
[0124] Mice are prepared, with 8 mice per group. Mice are purchased from Beijing Weitongda Biotechnology Co., Ltd., the strain is NPG (NOD-Prkdc.sup.scidll2rg.sup.null/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.
[0125] 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.
[0126] 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.
TABLE-US-00011 TABLE 11 Antibodies and 7-AAD Antibody name Manufacture Catalog number FITC anti-mouse CD45 Biolegend 103108 APC/Cy7 anti-human CD45 Biolegend 304014 Brilliant Violet 510 ™ anti-human CD3 Biolegend 300448 PE anti-human CD 19 Biolegend 363004 Brilliant Violet 421 ™ anti-human CD33 Biolegend 303416 APC anti-human CD56 Biolegend 304610 7-AAD Viability Staining Solution Biolegend 420404
[0127] 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.
TABLE-US-00012 TABLE 12 Number of Amount of Group cells Name of the added antibody the antibody NC 2 × 10{circumflex over ( )}5 — — Sample 2 × 10{circumflex over ( )}5 APC/Cy7 anti-human CD45 2 μl FITC anti-mouse CD45 2 μl
[0128] 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.
[0129] 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.
[0130] 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
[0131] 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.
TABLE-US-00013 TABLE 13 Group Number of cells Name of the added antibody Amount of the antibody NC 2 × 10{circumflex over ( )}5 Sample 2 × 10{circumflex over ( )}5 APC/Cy7 anti-human CD45 2 μl FITC anti-mouse CD45 2 μl Brilliant Violet 510 ™ anti-human CD3 2 μl PE anti-human CD 19 2 μl Brilliant Violet 421 ™ anti-human CD33 2 μl APC anti-human CD56 2 μl
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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
[0137] The results in
[0138] The results in