CN113577107A

CN113577107A - Preparation method and application of stem cells

Info

Publication number: CN113577107A
Application number: CN202110557156.XA
Authority: CN
Inventors: 施松涛
Original assignee: Individual
Current assignee: Sun Yat Sen University
Priority date: 2021-05-21
Filing date: 2021-05-21
Publication date: 2021-11-02

Abstract

The invention belongs to the field of biomedicine, and relates to a preparation method and application of stem cells. The present invention provides a method for preparing stem cells, the medium used for the stem cells is serum-free medium, the stem cells are dental pulp mesenchymal stem cells, and the stem cells obtained by the method are used to inhibit activated T cells and immune system diseases Treatment. The research of the present invention found that hMSC-DP obtained under serum-free medium culture has a stronger ability to induce T cell apoptosis than cells obtained under serum-cultured culture, and at the same time, serum-free cultured hMSC-DP is used for the treatment of DSS Induced acute colitis, it was found that the serum-free cultured hMSC-DP of the P5 and P10 generations had a strong therapeutic ability, suggesting that the serum-free cultured hMSC-DP has a strong potential for the treatment of immune diseases.

Description

Preparation method and application of stem cells

Technical Field

The invention belongs to the field of biological medicines, and relates to a preparation method and application of stem cells.

Background

Mesenchymal Stem Cells (MSCs) have been widely used clinically for the treatment of various diseases^[1]. MSCs can be isolated from various tissues, such as bone marrow, umbilical cord tissue, adipose tissue and dental pulp^[2]. International Society for Cell Therapy (ISCT)) The minimum standard for MSC application was established in 2006^[3]. However, no criteria have been established for quality assessment of MSCs from a particular organization.

Since the discovery of hMSC-DP^[4,5]Since then, their physiological and functional properties have been extensively studied^[6]. The excellent proliferative capacity, dentin/osteogenesis capacity and immunoregulatory capacity of hMSC-DP have been widely recognized^[6-8]. In recent years, hMSC-DP has been used in preclinical and clinical studies for tissue regeneration and immunotherapy^[7,9-13]. The quality and potency of MSCs is crucial to ensure their therapeutic effect^[14]. However, the MSC minimum criteria established by ISCT may not provide sufficient guidance for clinical use of hMSC-DP^[3]。

Previous studies have shown that MSCs cultured using Fetal Bovine Serum (FBS) may cause some ethical and immune response problems^[15-17]. Significant quality differences between FBS batches also limit the large-scale production of MSCs. Therefore, Serum (SF) free cultured MSCs are necessary for future clinical practice. FBS substitutes, including human platelet lysate and chemically-defined SF media, appear to provide sufficient nutritional support for culture expansion of hMSC-DP and maintain its tissue regeneration pluripotency^[18-20]. However, the detailed culture effect of SF medium on hMSC-DP and its cellular function are not fully understood.

Disclosure of Invention

In some embodiments, the present invention provides the use of dental pulp mesenchymal stem cells obtained by culturing the dental pulp mesenchymal stem cells in a serum-free medium for the preparation of a modulator of T cell activity.

In some embodiments, the number of passages of the dental pulp mesenchymal stem cells is less than or equal to passage 15.

In some embodiments, the number of passages of the dental pulp mesenchymal stem cells is less than or equal to passage 10.

In some embodiments, the number of passages of the dental pulp mesenchymal stem cells is less than or equal to passage 5.

In some embodiments, the dental pulp mesenchymal stem cells comprise DPSC and SHED.

In some embodiments, the modulator of T cell activity is a modulator of induction of T cell apoptosis.

In some embodiments, the dental pulp stem cells are used to treat an immune disease.

In some embodiments, the dental pulp stem cells are used to treat enteritis.

In some embodiments, the invention provides the use of dental pulp mesenchymal stem cells obtained by culturing the dental pulp mesenchymal stem cells in a serum-free medium for the preparation of a medicament for treating an immune disease.

In some embodiments, the present invention provides a method of preparing a stem cell using a serum-free medium, the stem cell being a dental pulp mesenchymal stem cell, the stem cell obtained by the method for modulating T cell activity.

In some embodiments, the stem cells obtained by the methods are used to induce apoptosis of T cells.

In some embodiments, the stem cells obtained by the methods are used to treat immune diseases.

In some embodiments, the stem cells obtained by the methods are used to treat enteritis.

In some embodiments, the present invention provides a modulator of T cell activity comprising dental pulp mesenchymal stem cells; the stem cells are obtained by using serum-free culture medium for culture.

In some embodiments, the modulator of T cell activity is for inducing apoptosis of a T cell.

In some embodiments, the modulator of T cell activity is used to treat an immune disease.

In some embodiments, the modulator of T cell activity is used to treat enteritis.

In some embodiments, the colitis is acute colitis.

In some embodiments, the T cell is an activated T cell.

The prior art shows that the stronger the ability to induce apoptosis of T cells, the greater the potential to treat immune diseases. The research of the invention finds that the MSC-DP obtained in the state of the serum-free culture medium integrally shows better capability of inducing T cell apoptosis (compared with the state of the serum culture), and particularly shows a result which is obviously superior to that in the state of the serum culture for stem cells with low generation number (better quality and potential).

In some embodiments, the serum-free medium comprises human platelet lysate.

In some embodiments, the serum-free medium further comprises DMEM.

In some embodiments, the serum-free medium further comprises L-glutamine or an L-glutamine analog.

In some embodiments, the L-glutamine analog is a GlutaMAX additive.

In some embodiments, the present invention provides a method for preparing stem cells using serum-free medium, wherein the stem cells are DPSCs, and wherein the stem cells obtained by the method are used for osteogenic differentiation.

At the P20 generation, the DPSC cells cultured in a serum-free state still have better osteogenic differentiation capacity, while the DPSC cells cultured in the serum lose the osteogenic differentiation capacity at the 20 th generation. Embodies that the DPSC under the serum-free culture condition can obtain and maintain stronger osteogenic differentiation capacity.

In some embodiments, the present studies have found that osteogenic differentiation can be achieved after a high passage number of DPSCs has been achieved, without serum.

In some embodiments, the invention provides a method for preparing stem cells, the culture medium used by the stem cells is serum-containing medium, the stem cells are SHED, and the stem cells obtained by the method are used for adipogenic differentiation.

In some embodiments, the number of SHEDs is greater than 15.

The research of the invention finds that for SHED, a serum culture medium is better selected during adipogenic differentiation, and the adipogenic differentiation capacity is obviously enhanced when the generation number reaches 20.

Drawings

FIG. 1 morphological features of hMSC-DP. a. Schematic representation of the isolation and culture of hMSC-DP under SE or SF culture conditions. The forms of hMSC-DP in P5, P10 and P20 generations under SE or SF culture conditions. Scale bar 50 μm. c. Morphological analysis of cells with high content imaging. hMSC-DP was co-stained with pharloid (orange), hoechst (blue) and WGA (green) at a scale bar of 100 μm, with n of 3 per group. d. Cell senescence assay, the percentage of SA- β -gal positive cells was calculated and compared between the groups P5, P10 and P20. Black arrows indicate positive staining of SA-. beta. -gal. Scale bar 100 μm. Each group n is 3-5. SE, serum culture; SF, serum free culture, bars are shown as the mean of each group. P <0.05, p <0.01, p < 0.001. ns, no significant difference.

FIG. 2 morphological features of hMSC-DP under SF culture conditions. a-b cell morphology analysis by high content imaging. Area comparison of nuclear (a) and cellular regions of DPSCs and SHEDs under SE and SF culture conditions (b). Each group n is 3. c. Cell senescence assay. The percentage of SA- β -gal positive cells was calculated and compared between SE and SF culture conditions. Each group n is 3-5. SE, serum; SF, serum free. The graph bar shows the average values of a-c. ns, no significant difference.

FIG. 3 proliferation potency and chromosomal stability of hMSC-DP at different generations. a. The colony forming ability of DPSCs and SHED under SE and SF culture conditions was evaluated by CFU-F analysis, and the number of clones of P5, P10 and P20 was calculated and compared, with n-3 per group. b the proliferative capacity of three different groups of donor-derived DPSCs and three different groups of donor-derived SHEDs was analyzed by population doubling assay. c proliferation rate of hMSC-DP was assessed by EdU staining. The percentage of EdU-positive cells was calculated for P5, P10 and P20 passages. d. Chromosome stability was assessed by karyotyping. The DPSCs and SHED karyotypes of P5, P10, and P20 generations were examined. SE, serum culture; SF, serum free culture. The bars are shown as the average of the groups. P <0.05, p <0.01, p < 0.001. ns, no significant difference.

FIG. 4 proliferation potency of hMSC-DP under SF culture conditions. CFU-F analysis. The number of hMSC-DP clones in SE or SF culture conditions was calculated and compared. Each group n is 3. b. Population doubling scores under SE and SF culture conditions in the DPSC and SHED groups were calculated and compared, respectively. Each group n is 3. Edu analysis. The percentage of EdU-positive cells between SE and SF culture conditions in the DPSC and SHED groups was calculated independently and compared. Each group n is 3. Scale bar 200 μm. SE, serum culture; SF, serum free culture. The bars are shown as the average of the groups. P < 0.05. ns, no significant difference.

FIG. 5 surface marker phenotype and in vitro immunomodulatory capacity of hMSC-DP at different passage numbers. a. MSC surface marker analysis of hMSC-DP at P5, P10 and P20 under SE and SF culture conditions was assessed by flow cytometry, showing a positive rate of CD34, CD45, CD73, CD90 and CD105, with n-3 per group. b. The percentage of CD146 positive cells at P5, P10 and P20 was calculated in DPSC and SHED, respectively. Each group n is 3. c to assess in vitro immunomodulatory capacity, hMSC-DP at P5, P10 and P20 were co-cultured with T cells and the percentage of apoptotic T cells was detected by flow cytometry. Each group n is 3. The bars are shown as the average of the groups. Comparison with control group: # p <0.05, # p <0.01, # p < 0.001. NS has no obvious difference with the control group. Comparison of hMSC-DP at P5, P10 and P20: p <0.05, p <0.01, p < 0.001. ns, no significant difference.

FIG. 6 surface marker phenotype and in vitro immunomodulatory capacity of hMSC-DP. Flow cytometry showed the percentage of CD146 positive hMSC-DP at P5, P10 and P20 under SE or SF culture conditions (a). The percentage of CD146 positive cells under SE and SF culture conditions was compared (b). Each group n is 3. c. The percentage of apoptotic T cells was calculated under SE and SF culture conditions. Each group n is 3. SE, serum; SF, serum free. The bars are shown as the average of the groups. P < 0.05. ns, no significant difference.

FIG. 7 multilineage differentiation of hMSC-DP. Osteogenic capacity of a-b hMSC-DP. (a) Comparison of the percentage of alizarin red staining at P5, P10 and P20. (b) Western blot analysis showed the expression levels of the osteogenic markers Runx2 and ALP. Each group n is 3. (c-d) lipidogenic capacity of hMSC-DP. (c) The percentage of oil red o-positive cells at P5, P10 and P20 passages was calculated and analyzed. (d) Western blot analysis showed the expression levels of adipogenic markers (including LPL and PPAR γ), with n-3 per group. e neurogenic differentiation of hMSC-DP. Immunofluorescent staining and Western blot analysis showed expression of the neural markers β iii-tubulin and NeuN. Scale bar 100 μm. SE, serum culture; SF, serum free culture. The bars are shown as the average of the groups. P <0.05, p <0.01, p < 0.001. ns, no significant difference.

FIG. 8 multilineage differentiation of hMSC-DP. a. Alizarin red staining method. The osteogenic capacity of P5, P10 and P20 were compared under SE and SF culture conditions. Each group n is 3. b. Oil red O staining assay. The lipid forming ability of hMSC-DP of P5, P10 and P20 under SE and SF culture conditions was compared. Each group n is 3. Se, serum-cultured at a scale of 50 μm; SF, serum free culture. The bars are shown as the average of the groups. P < 0.01. ns, no significant difference.

FIG. 9 immunotherapeutic Effect of hMSC-DP transplantation in Dextran Sodium Sulfate (DSS) -induced experimental colitis. a. Body weights were recorded daily for 8 days in both the DPSC and SHED transplant groups. The relative change in body weight was calculated. Each group n is 3. b. Disease Activity Index (DAI) was assessed on day 8 of DSS feeding. Each group n is 3. c. Colon length was measured on day 8. Each group n is 3. d. Histological scores based on H & E stained images were assessed on day 8. Histological scores were calculated for each group. Each group n is 3. SE, serum culture; SF, serum free culture. The bars are shown as the average of the groups. Comparison with untreated DSS group: # p <0.05, # p <0.01, # p < 0.001. NS, no significant difference from DSS group. Comparison of DPSCs and SHED for P5, P10 and P20: p <0.05, p <0.01, p < 0.001. ns, no significant difference.

FIG. 10 therapeutic effect of hMSC-DP on experimental colitis. a. Disease Activity Index (DAI) was compared between treatment groups of DPSCs and SHED between SE and SF culture conditions at P5, P10 and P20 generations. Each group n is 3. b. Colon length after treatment of P5, P10 and P20 passages DPSCs and SHED under SE and SF culture conditions was compared. Each group n is 3. c. Histological structures were examined by H & E staining. Histological scores of the groups were compared under SE and SF culture conditions. Each group n is 3. Scale bar 100 μm. SE, serum culture; SF, serum free culture. The bars are shown as the average of the groups. ns, no significant difference.

FIG. 11CD146 expression is associated with hMSC-DP proliferation, differentiation and immunomodulation. Western blot analysis showed expression of CD146 in P5, P10 and P20 in DPSC and SHED under SE or SF culture conditions. b. By Western blot analysis, scd 146 down-regulated the expression level of DPSCs and SHED for CD146 at P10 generation. c. Flow cytometry analysis showed a reduction in the percentage of CD146 positive cells after siCD146 treatment. Each group n is 3. EdU staining showed a decrease in the proliferation rate of hMSC-DP following siCD146 treatment. Each group n is 3. e. After coculture of siCD146 transfected hMSC-DP with T cells, the percentage of apoptotic T cells was reduced as assessed by flow cytometry. Each group n is 3. The expression of f-g knockdown CD146 reduced the osteogenic capacity of hMSC-DP, as assessed by the percentage of alizarin red staining (f) and Western blot for osteogenic markers (g). Each group n is 3. SE, serum culture; SF, serum free culture. The bars are shown as the average of the groups. P <0.01, p < 0.001. ns, no significant difference.

FIG. 12 the expression level of CD146 is related to hMSC-DP characteristics. Correlation of cd146 with each experimental parameter. b. The linear regression plots show the correlation between the expression level of CD146 and various experimental parameters, including CFU-F clones, percent EdU positive cells, percent alizarin red positive area, percent SA- β -gal positive cells, DAI score, HAI score and colon length in DSS-induced colitis treated by each group of cells.

FIG. 13CD146 controls the proliferation and immunomodulatory capacity of hMSC-DP through the ERK pathway. Western blot analysis showed expression levels of the ERK pathway in P5, P10 and P20 passages of DPSC and SHED under SE or SF culture conditions. b Western blot analysis showed that ERK pathways were down-regulated by siCD146 treatment in hMSC-DP. c ERK pathway inhibitor PD98059 inhibits the expression level of p-ERK. EdU staining showed a decrease in the proliferation rate of hMSC-DP after PD98059 treatment. Each group n is 3. e. As shown by flow cytometry, the percentage of apoptotic T cells decreased after co-culturing PD98059 treated hMSC-DP with T cells. Each group n is 3. The osteogenesis of hMSC-DP was examined after pd98059 treatment and was assessed by the percentage of alizarin red staining (f) and the protein expression level of the osteogenic marker (g). Each group n is 3. SE, serum culture; SF, serum free culture. The bars are shown as the average of the groups. P <0.05, p <0.01, p < 0.001. ns, no significant difference.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

List of abbreviations: hMSC-DP: human mesenchymal stem cells from dental pulp; and (4) DPSC: adult dental pulp stem cells; SHED: stem cells of deciduous teeth of a human being; and SE: serum; SF: serum-free; MSC: mesenchymal stem cells; ISCT: the international society for cell therapy; and (3) SOP: standard operating procedures; FBS: fetal bovine serum; EdU: 5-ethynyl-20-deoxyuridine; PD: multiplying the population; CFU: cloning a forming unit; WGA: wheat germ agglutinin; beta-gal: beta-galactosidase; SA- β -gal: senescence-associated beta-galactosidase enzyme; runx 2: runt-related transcription factor 2; ALP: alkaline phosphatase; PPAR γ: peroxisome proliferator activated receptor- γ 2; LPL: lipoprotein lipase; DSS: dextran sodium sulfate; DAI: an index of disease activity; HAI: histological activity index.

Materials and methods

1. Mouse

Male C57BL/6J mice were purchased from Zhongshan university, Guangzhou, China. All animal experiments were performed under agency approved protocols for animal studies (university of Zhongshan, SYSU-IACUC-2020-000394).

2. Isolated culture of hMSC-DP

Deciduous incisors and third molars, which were exfoliated by humans, were obtained from biological samples discarded in the oral hospital affiliated to Guanghua medical college, Zhongshan university.

All teeth were intact without any inflammation. The protocol (KQEC-2020-055-01) has been approved by the institutional medical Ethics Committee of the oral Hospital, university of Zhongshan. In the manner previously reported^[4,5]DPSCs and SHED were isolated and cultured until passage 2 (P2). hMSC-DP from P2 generation was cultured in the presence of Serum (SE) or in the absence of Serum (SF) until P20 generation.

Serum culture of hMSC-DP culture was performed in the previously reported manner^[4,5]. SF-cultured hMSC-DP was cultured in high glucose Dulbecco's modified Eagle Medium (DMEM, Invitrogen) supplemented with 5% volume fraction human platelet lysate (Compass biological), 100U/ml penicillin, 100mg/ml streptomycin (Gibco) and 1% volume fraction GlutaMAXTM supplement (Gibco). After the cells were confluent, they were digested and passaged by adding 0.25% trypsin (Gibco) containing 1mM EDTA and continuously cultured in SE or SF medium. At each passage, approximately 1X 10 was selected⁶hMSC-DP was seeded on 10cm dishes (Corning) and the excess cells were plated in serum-free cryopreservation medium (CELLBA)NKER^TM2) Storing in a-80 deg.C refrigerator. hMSC-DP from generations P5, P10 and P20 was selected for subsequent experiments.

3. Antibodies and reagents

anti-CD 34, CD45, CD73, CD90, CD105, CD146, and 7-AAD antibodies were purchased from BD Biosciences. anti-CD 3, CD8, and Annexin V antibodies were purchased from BioLegend. anti-Runx 2, ERK and p-ERK antibodies were purchased from Cell Signaling Technology. anti-CD 146, ALP, β III-tubulin and NeuN antibodies were purchased from Abcam. anti-LPL and β -actin antibodies were purchased from Invitrogen. anti-PPAR γ antibodies were purchased from Santa Cruz. WGA-488 was purchased from Biosharp. Hoechst 33342 was purchased from Sigma. The kFluor488-EDU cell proliferation assay kit was purchased from KeyGEN Biotech. Lipofectamine^TMRNAiMAX transfection reagent was purchased from Invitrogen. CD146 siRNA was purchased from rib Biotech. PD98059 was purchased from Beyotime. Cell senescence assay kits were purchased from Merck Millipore. Sodium dextran sulfate (DSS) was purchased from MP Biochemicals.

4. Flow cytometry analysis

For analysis of cell surface markers, 10⁶hMSC-DP suspensions were prepared at a density of 100. mu.l for the P5, P10 and P20 generations. Thereafter, 1. mu.l of the antibody was added to 100. mu.l of the cell suspension and incubated at 4 ℃ for 30 minutes in the dark, followed by using a flow cytometer (ACEA novoCyte)^TM) And (6) carrying out analysis. Apoptotic T cells were stained with CD3 and 7AAD antibodies for 30 min at 4 ℃, in the dark, washed twice with PBS, and then incubated with Annexin V antibody.

5. In vitro immunomodulatory capacity of hMSC-DP

Mixing P5, P10 and P20 (1X 10)⁵Per well) passage of hMSC-DP were seeded on 12-well culture plates (Corning) and incubated for 24 hours. T cells pre-stimulated by anti-CD 3 and CD8 antibodies were loaded directly onto hMSC-DP for 2 days of culture. Apoptotic T cell numbers were detected by flow cytometry.

6. Karyotyping analysis

Karyotyping of hMSC-DP, including chromosome number and G-band karyotype, was performed by Shanghai Haoge Biotechnology Inc. (Shanghai, China).

7. EdU dyeing method

Mixing hMSC-DP (2X 10 per well)⁴Individual cells) were seeded into 24-well plates (Corning) and cultured at 37 ℃ for 24 hours. Cells were treated with 200. mu.l of medium containing 50. mu.M of 5-ethynyl-20-deoxyuridine (EdU) for 2 hours. Cells were then detected using the EdU cell proliferation assay kit (KeyGEN, BioTECH) according to the manufacturer's instructions.

8. Population doubling assay

hMSC-DP generation P4 at 2X 10⁵The individual cells were seeded in 60mm dishes (Corning) containing SE or SF medium. When the cells reach about 70% -80% confluence, the cells are harvested and inoculated with the same number of cells into the culture medium. The population doubling rate (PD) was calculated by the following formula: PD ═ log2 (number of harvested cells/number of seeded cells). The total number of accumulated PD was determined from P5 to the time when cell division ceased. Each group was tested in duplicate with 3 independent cells from different sources.

9. Clone Forming Unit (CFU) assay

A total of 1,000 hMSC-DP per group were plated in 6cm dishes (Corning). After 10 days, cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet for 5 minutes. Clones containing 50 or more cells were selected for counting. This experiment was repeated three times.

10. Cellular morphological analysis by high content imaging

3,000 hMSC-DP of each group were seeded in 96-cell well plates (Corning) and cultured for 24 hours. After washing with PBS, fixation was performed for 10 minutes in 4% formaldehyde at room temperature. Cells were stained with Phalloidin, WGA and Hoechst 33342. The plates were scanned in an Operetta CLS and each group of cells was analyzed for cell size and shape using Harmony software (PerkinElmer).

11. Cellular senescence assay

Each group was given a total of 5X 10⁴Each hMSC-DP was inoculated in 24-well plates (Corning) and cultured for 24 hours. The cellular senescence assay was then performed according to the protocol provided by the cellular senescence assay kit (Merck Millipore).

12. Inducing osteogenesis, adipogenesis and neurogenesis differentiation

About 3X 10 per group⁵Individual hMSC-DP were cultured in 6cm plates until complete fusion. In the previously reported manner [21,22 ]]Osteogenic and adipogenic differentiation induction and evaluation were performed. To induce neuro-differentiation, a total of 4X 10 were used⁴The individual cells were seeded on a 24-well culture plate (Corning) and cultured for 24 hours. The cells were then replaced with neural differentiation medium and cultured for 10 days. Neural differentiation assessment was performed as previously reported^[5]。

13. siRNA transfection and chemical treatment

For siRNA transfection, hMSC-DP (5X 10)⁵Per well) were inoculated in 6-well plates in low serum medium (Opti-MEM, Gibco) and treated with CD146 siRNA (RIBO, China) or lipofectamine reagent control vector siRNA (invitrogen) for 24 hours according to the manufacturer's instructions. For chemical treatment, hMSC-DP was treated with 10. mu.M PD 98059. The treated cells were collected or used for further experiments.

14. hMSC-DP transplantation in acute colitis mice

Acute colitis was induced in mice on drinking water containing 3% DSS (MP biochemicals) for 8 days. On day 3 after feeding with DSS water, the total number of each group was 1X 10⁶One hMSC-DP was infused intravenously into colitis mice. All mice were euthanized and analyzed on day eight. Mice with induced colitis were evaluated as described previously^[23]。

15. Statistical analysis

GraphPad Prism 8 was used to perform statistical analysis. Comparisons between the two groups were analyzed using the independent student's two-tailed t-test, while comparisons between more than two groups were analyzed using one-way analysis of variance. P values <0.05 were considered statistically significant.

Example 1 morphological characterization of hMSC-DP

Experimental materials and methods are shown above in the materials and methods section.

A third molar from three donor sources was collected for DPSC isolation and a deciduous incisor from three donor sources was collected for SHED isolation. All donors were healthy volunteers, and teeth were free of caries and inflammation. According to the previous report^[4,5]From toothhMSC-DP, including DPSC and SHED, was isolated from the marrow. The P2 generation hMSC-DP was isolated and propagated in conventional serum-containing FBS (SE) or serum-free (SF) culture conditions with human platelet lysate to obtain the DPSC-SE, DPSC-SF, SHED-SE and SHED-SF groups (FIG. 1 a).

Cell morphology is considered an indicator of MSC function^[24]. hMSC-DP from all donors were adherent to petri dishes under SE and SF culture conditions, and P5 and P10 passages showed elongated spindle-shaped morphology. With serial passage to passage 20, hMSC-DP morphology became large, flat and irregular (fig. 1 b). To further analyze the morphological characteristics of the cells, we used high content imaging methods to analyze cell morphology, labeling the nucleus with hoechest, actin-labeled cytoskeleton and Wheat Germ Agglutinin (WGA) -labeled cell membrane^[25]. More than 500 cells were analyzed per group. In DPSC-SE (P)<0.01)，DPSC-SF(P<0.05)，SHED-SE(P<0.05) and SHED-SF (P)<0.01) group, the nucleus size of hMSC-DP at P20 generation was significantly larger than P5. The cell size of the P20 generation hMSC-DP also showed a tendency to become larger compared to that of P10 (FIG. 1 c). There was no significant difference in nuclear size between SE and SF culture conditions (FIG. 2a), but the cell size of SHED tended to decrease in SF culture compared to SE culture (P10 for P)<0.05, P20 time P<0.01). (FIG. 2 b).

Since cellular senescence may be associated with changes in cell size and nuclear size^[26]Therefore, we evaluated hMSC-DP for β -galactosidase (β -gal) positive senescing generations P5, P10, and P20. We found that the percentage of β -gal positive cells in each group increased from P5 to P20, and that this trend was statistically significant when comparing P5 to P20 (P20 in each group)<0.001) (fig. 1 d). However, there was no significant difference between SE and SF culture conditions (fig. 2 c). These data indicate that serial passage affects hMSC-DP characteristics, especially at P20.

Example 2 proliferative Capacity and chromosomal stability of hMSC-DP

Proliferation potency is an important parameter for assessing the function of MSCs. We inoculated hMSC-DP at a density of 1,000 cells/plate and cultured it for 10 days. The number of clones decreased from P5 to P20 (FIG. 3 a). Interestingly, SHED-SE showed higher clone numbers compared to the P20 generation SHED-SF group (FIG. 4 a). To examine the proliferative potential of hMSC-DP, population doubling scores were calculated from three donor-derived DPSCs and three donor-derived SHEDs. These cells showed similar proliferation capacity (FIG. 3b), with no significant difference under SE or SF culture conditions (FIG. 4 b). In addition, staining with 5-ethynyl-2' -deoxyuridine (EdU) showed a decrease in proliferation rate of hMSC-DP with serial passage, significantly lower at P20 (fig. 3c) compared to P5 (P < 0.001). SE and SF cultures did not differ in the EdU positivity rate (FIG. 4 c).

After culture amplification, it is crucial to use clinical grade human MSCs with normal genotype and chromosomal stability^[27]. We evaluated the karyotypes of P5, P10 and P20 passages DPSCs and SHED under SE and SF culture conditions. This is the same as the previous report that human MSCs maintain a relatively stable genome during culture amplification, and no chromosomal aberrations were detected in any of the test groups (fig. 3d)^[28,29]。

Example 3 surface phenotype and in vitro immunomodulatory Capacity of hMSC-DP

Next, we analyzed a series of stem cell surface markers, including those described by ISCT^[3]. hMSC-DPs from P5, P10 and P20 generations expressed the MSC surface markers CD73, CD90 and CD105, whereas they did not express CD34 and CD45 under SE and SF culture conditions (FIG. 5 a). Notably, the expression level of CD146 showed heterogeneity between groups (fig. 5a, fig. 6 a). We observed a gradual decrease in CD146 expression from P5 to P10 and from P10 to P20. CD146 expression at P20 was significantly reduced compared to P5 (DPSC-SE, DPSC-SF, SHED-SE and SHED-SF: P)<0.001) (fig. 5 b). Neither SE nor SF culture conditions showed an effect on CD146 expression levels (fig. 6 b). Previous studies have shown that CD146 is not only a melanoma cell adhesion molecule, but also a cell surface receptor for various ligands, involved in many physiological and pathological processes of cells^[30]. Of importanceThat is, CD146 is also expressed in MSCs.

To evaluate the in vitro immunomodulatory capacity of hMSC-DP, we co-cultured hMSC-DP with activated T cells for 2 days. Flow cytometry analysis showed that the hMSC-DP of P5 and P10 passages were able to significantly induce the level of T cell apoptosis compared to the control group, but the hMSC-DP of P20 lost the ability to induce T cell apoptosis (FIG. 5 c). hMSC-DP of P5 and P10 showed higher immunomodulatory capacity under SF culture compared to SE cultured cells. DPSCs of P5 passage showed significantly reduced immunomodulatory capacity (P <0.05) (fig. 6 c). These results indicate that hMSC-DP cultured in SF medium may be more suitable for immunotherapy.

Example 4 multilineage differentiation of hMSC-DP

The multi-lineage differentiation potential is a key parameter for assessing MSC function. To evaluate the osteogenic capacity of hMSC-DP, we cultured hMSC-DP for two weeks at generations P5, P10 and P20 in SE and SF osteogenic induction medium. Alizarin red staining showed that hMSC-DP showed a reduced tendency to mineralized nodule formation from P5 to P20 passages, indicating that continued passaging impaired the osteogenic differentiation capacity of hMSC-DP (fig. 7a, fig. 8 a). Western blot analysis confirmed that serial passage reduced the expression levels of run-associated transcription factor 2(Runx2) and alkaline phosphatase (ALP) (FIG. 7 b). Interestingly, P20 passage SHED still had a relatively strong osteogenic capacity under SF culture compared to SE cultured DPSCs, whereas P20 passage DPSCs had a significantly stronger capacity to form mineralized nodules under SF culture. All these results indicate that SF medium has an advantage in maintaining the osteogenic ability of hMSC-DP.

Our previous studies showed that hMSC-DP was able to differentiate into adipocytes, but with limited capacity^[4,5]. Here, we show that hMSC-DP from P5 to P20 passages showed limited adipogenic differentiation capacity after 6 weeks of adipogenic induction in SE or SF medium (FIG. 7c, FIG. 8 b). Western blot analysis confirmed the expression of two adipocyte-specific marker proteins in each group, peroxisome proliferator-activated receptor- γ 2(PPAR γ) and lipoprotein lipase (LPL) (fig. 7 d).

Previous studies have shown that DPSCs and SHEDs are capable of differentiating into neural cells, presumably because they originate from the neural crest^[31-33]. hMSC-DP maintained neuro-differentiation ability from P5 to P20 under SE or SF culture conditions, and expression of the neuronal markers β III tubulin and NeuN was also demonstrated by Western blot and immunostaining evaluation (fig. 7 e).

Example 5hMSC-DP demonstrates immunotherapeutic Effect in Dextran Sodium Sulfate (DSS) -induced experimental colitis

DSS-induced experimental colitis has been widely used to assess the in vivo immunomodulatory capacity of mouse MSCs^[23,34]. To further evaluate the immunomodulatory capacity of hMSC-DP, we injected SE and SF cultured P5, P10 and P20 passages of hMSC-DP into experimental colitis mice by tail vein infusion on day 3 after induction of 3% DSS. Experimental colitis mice were sacrificed 8 days after DSS induction. It was observed at day 8 that DPSC-SE of P5 generation and all DPSCs cultured under SF improved the body weight of the mice; wherein with P10 (P)<0.05) and P20 (P)<0.05) cells, better body weight recovery (P) was achieved with the P5 generation of DPSC-SF<0.01). The SHED-SF of P20 lost its ability to improve body weight to some extent (FIG. 9 a). Treatment with hMSC-DP at generation P5 significantly improved the Disease Activity Index (DAI) compared to the untreated DSS group, whereas hMSC-DP at generation P20 did not improve DAI (FIG. 9 b). Anatomical images of each group of colons showed that both DPSC-SE and SF at P5 had therapeutic effect on DSS-induced colitis; SHED-SE in P5 and SHED-SF in P5 and P10 improved colon length compared to untreated group (FIG. 9c, FIG. 10 b). Histological scoring of HE stained images showed that except DPSC-SE and SHED at P20, hMSC-DP of the other groups had therapeutic effect on DSS-induced colitis (fig. 9d, fig. 10 c). In general, the immunoregulatory capacity of stem cells gradually decreases with continued passage: in experimental colitis mice, the therapeutic effect of hMSC-DP at P10 was weaker than that of hMSC-DP at P5, while hMSC-DP at P20 showed little therapeutic effect. The DPSC cultured by SF shows stronger immunity on the whole than the DPSC cultured by SEModulation (fig. 10).

Example 6 functional surface molecule CD146 prediction of hMSC-DP function

CD146 is one of the surface markers of hMSC-DP^[35]. Previous studies have shown that MSCs with high CD146 expression have better osteogenic and immunomodulatory capacity than MSCs with low CD146 expression^[36-38]. We found that the expression level of CD146 in hMSC-DP decreased with passage (FIG. 11 a). To further explore the role of CD146 in hMSC-DP, we blocked the expression of CD146 in hMSC-DP with siCD146 as shown by Western blotting and flow cytometry (FIG. 11b, c). Following siCD146 interference, EdU staining, T cell apoptosis rate, alizarin red staining, and Western blot analysis of ALP and Runx2 demonstrated significant decreases in proliferation, in vitro immunomodulation, and osteogenic capacity (FIG. 11 d-g). Furthermore, we analyzed the correlation between these potency indices and CD146 expression levels and found that the potency of hMSC-DP was positively correlated with CD146 expression levels, particularly in terms of proliferation, osteogenesis and in vivo immunoregulatory capacity (fig. 12). These results indicate that CD146 is a functional surface marker, reflecting the function of hMSC-DP.

It is well known that the ERK/p-ERK pathway plays a key role in the regulation of MSC biological behavior^[22,39,40]. We found that the expression level of p-ERK, but not ERK, was consistent with the level of CD146 in hMSC-DP (FIG. 13 a). Western blot results showed that siCD146 treatment down-regulated the expression of p-ERK in hMSC-DP (FIG. 13b), indicating that CD146 may maintain hMSC-DP function through the ERK/p-ERK pathway. To validate this hypothesis, we used the ERK inhibitor PD98059 to reduce the expression level of pERK in hMSC-DP as assessed by Western blot experiments (FIG. 13 c). As shown by EdU staining and T cell apoptosis rate, we found that it significantly reduced proliferation rate and immunosuppressive capacity (fig. 13d, e). Interestingly, osteogenic differentiation was not affected in the DPSCs group but increased in the SHED group after treatment with p-ERK inhibitors (FIG. 13f, g)^[22,41]. These results indicate that CD146 can be maintained fine by the ERK pathway in hMSC-DPCell proliferation and immunoregulatory ability, but not the osteogenic effect.

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Claims

1. The application of dental pulp stem cells in the preparation of T cell activity regulators or the treatment of immune diseases, wherein the dental pulp mesenchymal stem cells are obtained by culturing in a serum-free medium.

2. The application according to claim 1, wherein the generation number of the dental pulp mesenchymal stem cells is less than or equal to the 15th generation;

Preferably, the generation number of the dental pulp mesenchymal stem cells is less than or equal to the 10th generation;

Preferably, the passage number of the dental pulp mesenchymal stem cells is less than or equal to the fifth passage.

3. application as claimed in claim 1, is characterized in that, described dental pulp mesenchymal stem cell is DPSC or SHED;

Preferably, the T cell activity regulator is a T cell activity inhibitor;

Preferably, the T cells are activated T cells;

Preferably, the immune disease is enteritis.

4. The use according to claim 3, wherein the enteritis is acute colitis.

5. A method for preparing stem cells, wherein the culture medium used by the stem cells is a serum-free medium, the stem cells are dental pulp mesenchymal stem cells, and the stem cells obtained by the method are used to regulate T cell activity;

Preferably, the stem cells obtained by the method are used to induce apoptosis of T cells;

Preferably, the T cells are activated T cells.

6. The method of claim 5, wherein the stem cells obtained by the method are used for the treatment of immune diseases;

Preferably, the stem cells obtained by the method are used for the treatment of enteritis;

Preferably, the enteritis is acute colitis.

7. The method of claim 5, wherein the generation number of the dental pulp mesenchymal stem cells is less than or equal to the 15th generation;

Preferably, the generation of the dental pulp mesenchymal stem cells is less than or equal to the fifth generation;

Preferably, the dental pulp mesenchymal stem cells are DPSCs or SHEDs.

8. A T cell activity regulator, characterized in that the regulator contains dental pulp mesenchymal stem cells; the stem cells are obtained by culturing in a serum-free medium;

Preferably, the generation number of the dental pulp mesenchymal stem cells is less than or equal to the 15th generation;

9. The T cell activity regulator of claim 8, wherein the T cell activity regulator is used to induce apoptosis of T cells;

Preferably, the T cells are activated T cells;

Preferably, the T cell activity modulator is used for the treatment of immune diseases;

Preferably, the T cell activity modulator is used to treat enteritis;

Preferably, the enteritis is acute colitis.

10. The use according to any one of claims 1-4, or the method according to any one of claims 5-7, or the T cell activity regulator according to any one of claims 8-9, wherein the The serum-free medium contains human platelet lysate;

Preferably, the serum-free medium further contains DMEM;

Preferably, the serum-free medium further contains L-glutamine or L-glutamine analogs;

Preferably, the L-glutamine analog is a GlutaMAX additive.