Small molecular Stemazole for promoting stem cell clonal formation and applications thereof

Abstract

Provided in the present invention are uses of a compound as represented by formula I in preparing a stem cell apoptosis antagonist. Also provided are uses of the compound as represented by formula I in preparing a medicament for preventing or treating cell apoptosis-related diseases, and a culturing method employing the compound as represented by formula I for stem cell culturing. The compound as represented by formula I is (I). ##STR00001##

Claims

1. A method of culturing stem cells, including: performing in vitro single-cell culture in the presence of a compound as represented by formula I: ##STR00006## wherein in formula I, R.sub.1 is H, halogen, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxyl; R.sub.2 is H, or C.sub.1-C.sub.4 alkyl; R.sub.3 is H, halogen, sulfhydryl, hydroxy, or C.sub.1-C.sub.4 alkyl, or heteroalkyl in which a heteroatom is oxygen or sulfur, and characterized in that, a cell concentration of the in vitro single-cell culture is 1-100 cells/0.1 mL or 10-10.sup.3 cells/mL.

2. The method according to claim 1, characterized in that, the stem cells are human stem cells.

3. The method according to claim 1, characterized in that, the method includes: adding the compound as represented by formula I into culture system of the in vitro single-cell culture, wherein a concentration of the compound as represented by formula I in the culture system is 1-100 μM.

4. The method according to claim 1, characterized in that, R.sub.1 is Br.

5. The method according to claim 1, characterized in that, the heteroalkyl is C1-C4 alkyl substituted by oxygen or sulfur.

6. The method according to claim 1, characterized in that, the compound as represented by formula I is compound 4-(4-(5-mercapto-1,3,4-oxadiazole-2-yl)phenyl)thiosemicarbazide as represented by formula Ia: ##STR00007## or, the compound as represented by formula I is compound 4-(2-Br-4-(5-mercapto-1,3,4-oxadiazole-2-yl)phenyl)thiosemicarbazide as represented by formula Ib: ##STR00008##

7. The method according to claim 1, characterized in that, the stein cells are human embryonic stein cells, human somatic stein cells or induced pluripotent stein cells.

8. The method according to claim 1, characterized in that, the stein cells are human embryonic stein cell strain H9, human somatic neural stein cells and/or human somatic pancreatic stem cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

(2) FIG. 1 shows the staining results of the cells in the experiment group and the control group in Example 1, in which FIG. 1A shows the results of alkaline phosphatase staining of the experiment group (ST) and the control group (DMSO) on day 4 and day 8, and FIG. 1B shows the results of the OCT-4 immunofluorescence staining of the experiment group (ST) on day 1 and day 8.

(3) FIG. 2 shows the changes in the number of cells in the three groups in Example 4 as a function of culture time.

(4) FIG. 3 shows the expression of important markers of human embryonic stem cells in Example 5, in which FIG. 3A shows the detection by quantitative PCR and FIG. 3B shows the detection by immunofluorescence staining.

(5) FIG. 4 shows the expression of proliferation markers of human embryonic stem cell in Example 5, in which FIG. 4A shows the results of immunofluorescence staining and FIG. 4B shows the positive rate of expression of proliferation markers.

(6) FIG. 5 shows the differentiation ability to form embryoid bodies (EBs) in Example 5, in which FIG. 5A is a microscopic image of each group at different time and FIGS. 5B and 5C show the diameters and number of the formed EBs, respectively.

(7) FIG. 6 shows the cell viability of human hippocampal neurosphere stem cells in the presence of Stemazole and Br-Stemazole in Example 6.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(8) The present invention is described below with reference to particular Examples. Those skilled in the art can understand that the Examples are only used to illustrate the invention and are not intended to limit the scope of the invention in any way.

(9) Pharmaceutical materials, reagent materials and the like used in the following Examples are commercially available products, unless particularly stated. Some of the materials are purchased as follows:

(10) Human embryonic stein cell strain H9 is purchased from Peking University Stem Cell Center.

(11) Specific apoptosis detection kit Annexin V/PI is purchased from Beyotime Biotechnology, China.

(12) Experimental methods in the following Examples are conventional methods, unless particularly stated. Some of the experimental methods are as follows:

(13) Alkaline Phosphatase Staining—

(14) 1) Rinsing cells with PBS, 3 times;

(15) 2) Fixing the cell with 4% paraformaldehyde for 10 minutes;

(16) 3) Washing with PBS, 3 times;

(17) 4) Preparing alkaline phosphatase staining agent:solution A:solution B:solution C=10 μl:10 μl:1 ml;

(18) 5) Adding the alkaline phosphatase staining agent to cover the cells, and staining for 20 minutes at room temperature in dark;

(19) 6) Aspirating off the staining agent, rinsing with PBS 3 times, storing the cells in PBS, and observing staining under a microscope.

(20) Immunofluorescence Staining—

(21) 1) Washing processed slide with cells thereon with 1×PBS 3 times, 5 minutes for each time;

(22) 2) permeabilizing the cells with PBS containing 0.5% Triton X-100 at room temperature for 15 minutes;

(23) 3) Washing with PBS 3 times, 5 minutes for each time;

(24) 4) Blocking with 5% sheep serum at 37° C. for 30 minutes;

(25) 5) Adding primary antibody and incubating at room temperature for 3-4 hours, or at 4° C. overnight;

(26) 6) Washing with PBS 3 times, 5 minutes for each time;

(27) 7) Adding fluorescent-labeled secondary antibody, and incubating at 37° C. for 1 hour;

(28) 8) Washing with PBS 3 times, 5 minutes for each time;

(29) 9) Mounting the slide with mounting agent containing DAPI;

(30) 10) Observing under a fluorescence microscope or confocal microscope, and taking photos.

(31) Complete culture medium used in the following Examples is:

(32) Basic medium DMEM/F12, supplemented with 20% serum substitute, 2 mM glutamine, 0.1 mM 3-thioethanol, 0.01 mM non-essential amino acid, 100 units/ml penicillin, 100 units/ml streptomycin, and 10 ng/ml growth factor bFGF.

Example 1 Stemazole Promotes the Formation of Single Cell Clones in Single-Cell Culture of Human Embryonic Stem Cells

(33) After being digested into single cells, human embryonic stem cell strain H9 cells were divided into two groups as follows, and inoculated on low density MEF feeder layer cells in 96-well plates:

(34) Experiment group (ST group): complete medium+Stemazole dissolved in DMSO at a final concentration in the culture medium of 100 μM;

(35) Control group (DMSO group): complete medium+DMSO with an amount corresponding to the experiment group.

(36) The cell ratio of each well in the two groups was H9 cell/100 MEF cells, and the medium was supplemented once every 4 days.

(37) Alkaline phosphatase staining was performed on day 4 and day 8. The results are shown in FIG. 1A: the number and sizes of the clones formed by the H9 cells in the DMSO group were less and smaller, while the number and sizes of the clones formed in ST group were higher and larger. Statistical analysis of the number of cell clones in the two groups showed that the number of clones of alkaline phosphatase positive cells in the DMSO group averaged about 8 on day 4, and the number of clones of the alkaline phosphatase positive cells in the ST group averaged about 18, and the difference between the numbers of clones in the two groups was significant. The number of clones of alkaline phosphatase positive cells in the DMSO group averaged about 12 on day 8, and the number of clones of the alkaline phosphatase positive cells in the ST group averaged about 23, and the difference between the number of the clones in the two groups was significant. As to the size, the diameter of the alkaline phosphatase positive cells in the DMSO group on day 4 averaged about 50 microns, and the diameter of the alkaline phosphatase positive cells in the ST group averaged about 100 microns. There was no significant difference between the sizes of the clones in the two groups. The diameter of the alkaline phosphatase positive cells in the DMSO group on day 8 averaged about 70 microns, and the diameter of the alkaline phosphatase positive cells in the ST group averaged about 200 microns. There was a significant difference between the sizes of the clones in the two groups.

(38) OCT-4 immunofluorescence staining was performed on day 1 and day 8. The results are shown in FIG. 1B: in the ST group, single 1-19 cells were observed growing by adhering to the wall, which revealed that H1-9 single cells had grown into monoclonal cells; and in the DMSO group, no single 1-19 cells were observed adherent to the wall, neither 1-19 monoclonal cells were found.

Example 2 Stemazole Reduces Apoptosis in Human Embryonic Stem Cell Single-Cell Culture

(39) Human embryonic stem cell line H9 cells were digested into single cells, divided into 3 groups as follows, and then inoculated at a low density on low-density MEF feeder layer cells in 6-well plates:

(40) Experiment group (ST group): complete medium+Stemazole dissolved in DMSO at a concentration in the final culture medium of 100 μM;

(41) Negative control group (DMSO group): complete medium+DMSO at an amount corresponding to the experiment group;

(42) Positive control group (Y27632 group): complete medium+Y27632 dissolved in DMSO at a concentration in the final culture medium of 10 μM.

(43) The cell ratio of each well of the three groups was 10 H9 cells/1 MEF cell.

(44) Apoptosis was detected by flow cytometry using the specific apoptosis assay kit Annexin V/PI on day 2 of inoculation.

(45) The results showed that: the DMSO group had the largest number of Annexin V positive apoptotic cells, which accounted for 54.1% in average; apoptotic cells in the ST group were obviously less than that in the DMSO group, and accounted for 25.2% in average; and the apoptotic cells in the Y27632 group was the least, and accounted for 20.1% in average. Statistical analysis showed that there was a significant difference between the DMSO group and the ST group as well as between the DMSO group and the Y27632 group, but there was no significant difference between the ST group and the Y27632 group.

(46) The small molecule compound Stemazole of the present invention has a completely different chemical structure from that of the ROCK inhibitor Y-27632, but achieves a comparable inhibitory effect on apoptosis. Thus it can be used as a new antagonist of apoptosis of stem cells.

Example 3 Stemazole Reduces Apoptosis of Human Embryonic Stem Cells During Total Nutrient Removal Culture

(47) After passaged, human embryonic stem cell line H9 cells were divided into the following two groups and inoculated in 6-well plates:

(48) Experiment group (ST group): total nutrient removal culture medium (complete medium with removal of serum and growth factor)+Stemazole dissolved in DMSO at a final concentration in the culture medium of 100 μM;

(49) Control group (DMSO group): total nutrient removal culture medium+DMSO with an amount corresponding to the experiment group;

(50) Normal culture group: complete medium.

(51) Apoptosis was detected by flow cytometry using the specific apoptosis assay kit Annexin V/PI.

(52) The results showed that: the percent apoptotic cells in the normal culture group was about 0.4%; while, under the condition of any exogenous serum or growth factor being removed (i.e., under the condition of total nutrient removal or nutrient starving) for 15 hours, early apoptosis occurred in the DMSO group, and the apoptosis rate was about 43%. In contrast, there was no significant apoptosis in the ST group, in which the apoptosis rate was about 8%. Compared to the DMSO group, an obvious anti-apoptotic performance was found in the ST group.

Example 4 Stemazole Reduces Apoptosis Rather than Promotes Proliferation in Single-Cell Culture of Human Embryonic Stem Cells

(53) Human embryonic stem cell strain H1-9 cells were digested into single cells, then divided into following three groups and inoculated on low-density MEF feeder layer cells in 6-well plates:

(54) Experiment group (ST group): complete medium+Stemazole dissolved in DMSO at a final concentration in the culture medium of 100 μM;

(55) Control group (DMSO group): complete medium+DMSO with the amount corresponding to the experiment group;

(56) Normal culture group: complete medium.

(57) The cells in each well of the three groups were maintained well grown on the MEF feeder layer cells, and the medium was supplemented once every 4 days.

(58) The number of cells in the three groups was measured as a function of time, and the results are shown in FIG. 2: the number of the cells in the ST experiment group did not increase, and after a slight decrease, remained constant as time passed. The results of this experiment prove that the biological effect of ST is to maintain cell survival, and ST does not show its activity to promote proliferation of the H9 cell line.

Example 5 Stemazole Maintains the Stemness of Human Embryonic Stem Cells

(59) I. Expression of Important Marker Proteins of Human Embryonic Stem Cells

(60) Following the experimental method in Example 3, H9 cells were divided into experiment group (ST group) and control group (DMSO group) and cultured. After 15 hours of total nutrient removal culture, the expression of each of human embryonic stem cells' important markers OCT-4, Sox2, FDF4, KLF4, REX1, and Nanog was detected by quantitative PCR.

(61) TABLE-US-00001 1. Quantitative PCR reaction system: Agent Volume 2X SYBR Green Mix 7.5 μl Upstream primer 0.5 μl Downstream primer 0.5 μl cDNA template   1 μl Sterile water 5.5 μl

(62) TABLE-US-00002 2. Reaction conditions of quantitative PCR: Step Setting Stage I 95° C., 5 min Stage II 95° C., 15 s; 60° C., 1 min; 40 cycles Stage III 95° C., 15 s; 60° C., 1 min; 95° C., 30 s; 1 cycle
3. Data Analysis:

(63) Ct (Cycle threshold) of each sample was measured by calculating 2.sup.−.sup.ΔΔ.sup.Ct, to compare differential expression of specific gene between different samples. In order to reduce systematic error and sampling error, triplicate wells in parallel were set for each sample. The average value of the triplicate wells was taken as the Ct of the sample, and then 2.sup.−.sup.ΔΔ.sup.Ct was calculated according to the formulas below. Each experiment was repeated at least 3 times, and the results are shown in a column chart in form of average value±standard deviation.
ΔCt.sub.experiment group=Ct.sub.gene in experiment group−Ct.sub.reference gene in experiment group
ΔΔCt.sub.control group=Ct.sub.gene in control group−Ct.sub.reference gene in control group
ΔΔCt=ΔCt.sub.experiment group−ΔCt.sub.control group
2.sup.−.sup.ΔΔ.sup.Ct=2.sup.−(.sup.Δ.sup.Ctexperiment group−.sup.Δ.sup.Ctcontrol group)

(64) The results are shown in FIG. 3A. It was found that after being subjected to the nutrient starving culture, the H9 cells still expressed important markers OCT-4, Sox2, FDF4, KLF4, REX1, and Nanog of stem cells, i.e., Stemazole maintained the stem cell characteristics of the 1-19 cells under the condition of total nutrient removal culture.

(65) In addition, still following the experimental method in Example 3, 1H9 cells were divided into experiment group (ST group), control group (DMSO group) and normal culture group and cultured. After total nutrient removal culture for 15 hours, the expression of each of human embryonic stem cells' important markers OCT-4 and E-Cadherin was detected by immunofluorescence staining.

(66) The results are shown in FIG. 3B. It was found that after being subjected to the nutrient starving culture, the H9 cells still expressed important markers OCT-4 and E-Cadherin of stem cells, i.e., Stemazole maintained the stem cell characteristics of 1-19 cells under the condition of total nutrient removal culture.

(67) II. Detection of Proliferation Marker of Human Embryonic Stem Cells

(68) Following the experimental method in Example 3, H9 cells were divided into experiment group (ST group), control group (DMSO group) and normal culture group and cultured. After total nutrient removal culture for 15 hours, immunofluorescence staining of the H9 cells was conducted for Ki67 (a proliferation marker).

(69) The results are shown in FIG. 4A and FIG. 4B. It was found that the H9 cells still highly expressed Ki67, i.e., Stemazole maintained the proliferation ability of the H9 cells.

(70) III. Detection of Differentiation Potential for Forming Embryoid Bodies

(71) Following the experimental method in Example 3, H9 cells were divided into experiment group (ST group), control group (DMSO group) and normal culture group and cultured. The formation of EBs was observed under a microscope at the 15th hour, and on day 2 and day 4 of total nutrient removal culture respectively, and the diameter and number of EBs were measured at the 15th hours.

(72) The results are shown in FIGS. 5A, 5B and 5C. It was found that EBs could still be formed from the H9 cells, i.e., Stemazole maintained the multi-directional differentiation potential of the H9 cells. And compared to the unprotected DMSO group, more EBs with larger diameter were formed in the ST group which was protected by Stemazole, and there was a significant difference between the two groups.

(73) IV. Detection of Ability of Induced Differentiation into Endoderm, Mesoderm and Ectoderm Cells

(74) Following the experimental method in Example 3, H9 cells were divided into experiment group (ST group), control group (DMSO group) and normal culture group and cultured.

(75) The H9 cells were digested by Collagenase IV, washed with DMEM (high glucose) 3 times, and gently blown into small cell clusters, then were inoculated into 100 mm petri dishes with the culture medium changed into differentiation culture medium (DMEM/F12, 20% FBS, 1× non-essential amino acid, 1×sodium pyruvate, 1×β-mercaptoethanol, double antibiotics, and 2 mM glutamine). The medium was changed every 2 days, and the dishes were observed on each day. After 4 days, RNA was extracted and the differentiation ability of the H9 cells to endoderm, mesoderm and ectoderm cells was detected.

(76) The results showed that the EBs formed from the cells in the three groups multi-directionally differentiated. The expression of CK8 and CK18 of endoderm; Brachyury and MSX1 of mesoderm; and GFAP, Pax6, and MAP2 of ectoderm were all detected. In terms of the expression of mesoderm marker MSX1, the expression of the DMSO group and the expression of the ST group (both groups were subjected to nutrient starving for 15 hours) were higher than that of normal control group cells which did not experience nutrient starving, and the expression of the DMSO group was comparable to that of the ST group. In terms of expression of ectoderm marker GFAP, the expression of the DMSO group which was subjected to nutrient starving for 15 hours was less than that of the ST group, and the expression of the ST group was comparable to that of the normal control group cells which did not experience nutrient starving. In terms of expression of ectoderm marker Pax6, the expression of the DMSO group was comparable to that of the ST group, while both higher than that of normal control group cells which did not experience nutrient starving. There was no significant difference found in the expression of other individual markers. All experiments had been repeated 3 times to confirm the results.

Example 6 Stemazole and Br-Stemazole Improve Cell Viability of Human Hippocampal Neurosphere Stem Cells

(77) 1. Culture of Human Hippocampal Neurosphere Stem Cells

(78) 1) On a superclean bench and under sterile conditions, human embryo hippocampus was taken out;

(79) 2) After envelope and blood vessels were removed under a dissecting microscope, tissue obtained was rinsed with pre-cooled PBS at 4° C. 3-5 times, leaving a very small amount of PBS;

(80) 3) The tissue was cut into pieces using ophthalmic scissors, and about 5 ml of culture medium was added to suspend the tissue pieces, and then the obtained cell suspension was gently pipetted to achieve single cell state with a pipette tip;

(81) 4) The cell suspension was filtered through a 40 μm pore size cell sieve;

(82) 5) Cell density was regulated to 1×10.sup.6/ml, and then the cells were inoculated into a 75 cm.sup.3 glass culture flask;

(83) 6) The cells in the flask were cultured in a 37° C., 5% CO.sub.2 cell culture incubator.

(84) 2. Cell Viability Assay

(85) The human hippocampal neurosphere stem cells were mechanically pipetted into small clones, regulated into a suitable density, and inoculated into a 96-well, black-wall plate at about 50 neurospheres/110 μL cell suspension/well. Solvent DMSO was used as control, and series concentrations of compound preparations were added respectively at 10 μL/well, in which the compound was Stemazole or Br-Stemazole, thereby obtaining concentrations of the compound in the cell suspensions as shown in FIG. 6; then the plate was cultured in a 37° C., 5% CO.sub.2 cell culture incubator. Without changing the medium, ATP was added at 20 μL/well after the compound acted for 4 days or other time, and the viability of cells was detected.

(86) The results are shown in FIG. 6. It was found that on screening platform of human neurosphere stem cells derived from hippocampus, both compound Stemazole and Br-Stemazole had a protective effect on the cell viability of human hippocampal neurosphere stem cells, in a dose-effect relationship. Compared with Br-Stemazole, the biological activity of Stemazole was stronger, and the cell viability increased up to 2.7 times over the control group; while Br-Stemazole also was found to have a biological activity, and the cell viability increased up to 2.1 times over the control group.

(87) Human SSP neural precursor cells and human embryonic pancreatic precursor cells were also used for performing the experiment, and the same results were obtained.

(88) In summary, the present application has demonstrated that the application of the compounds of formula I to in vitro single-cell culture of stem cells can significantly promote the formation of single cell clones of stem cells, and can also significantly inhibit apoptosis of stein cells in low concentration culture. Therefore, the compounds can help to solve technical problems in cell isolation and culture. Moreover, the compounds of formula I can be used for in vitro total nutrient removal culture of stem cells, during which they can significantly inhibit apoptosis of the stem cells in the absence of nutrients such as serum, growth factor and the like essential to in vitro culture, thereby excluding the contamination and influence of exogenous genes or proteins on stem cell growth, which is more beneficial to basic research and clinical application of stem cells. In particular, the use of the compounds of formula I also maintains the characteristics, proliferative capacity and multi-directional differentiation potential of stem cells, because the compounds exert their anti-apoptotic effect without sacrificing stem cell characteristics.

(89) In addition, the compounds of formula I can also be used for somatic stem cells and can increase the cell viability of human somatic stem cells such as human hippocampal neurosphere stem cells, human SSP neural precursor cells, human pancreatic precursor cells and the like.

(90) In conclusion, the compounds of formula I can be widely applied to in vitro culture of human cells, especially human embryonic stem cells or human somatic stem cells, and lay a foundation for further deeper research.

(91) The above description of the specific embodiments of the present invention is not intended to limit the invention, and various modifications and changes may be made by those skilled in the art without departing from the spirit and scope of the invention, and belong to the scope of the following claims of the invention.