CN111471648A

CN111471648A - Preparation method of pretreated and activated mesenchymal stem cells and application of pretreated and activated mesenchymal stem cells in treatment of myeloid leukemia

Info

Publication number: CN111471648A
Application number: CN202010282962.6A
Authority: CN
Inventors: 沈萍萍; 孙璐琛; 杨南飞; 郑薇; 左诗曼
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2020-04-10
Filing date: 2020-04-10
Publication date: 2020-07-31
Also published as: CN113512525A

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to application of umbilical cord-derived mesenchymal stem cells to differentiation therapy of acute and chronic myelogenous leukemia, wherein the mesenchymal stem cells are human-derived umbilical cord mesenchymal stem cells (UC-MSCs), and the mesenchymal stem cells (Active MSCs) are obtained after pretreatment and activation of specific chemical small molecules-1, 25D3(1, 2-Dihydroxyvitamin D3, 1, 25-dihydrovitamin D3), can efficiently induce differentiation of AM 5 cells, and are realized by secreting interleukin 6(I L-6) and indole-2, 3 dioxygenase (IDO), are allogeneic mesenchymal stem cells, and can remarkably improve the expression and secretion capacities of I L-6 and IDO after treatment and activation of VDR agonist 1, 25D3, remarkably enhance the anti-transplantation capacity, remarkably enhance the mesenchymal stem cells, and provide clinical treatment for mesenchymal stem cells without toxic and side effects, and the mesenchymal stem cell transplantation therapy method is a clinical treatment method for leukemia with low toxic and chemotherapy-free from residual drugs, and the leukemia is a clinical treatment idea of leukemia.

Description

Preparation method of pretreated and activated mesenchymal stem cells and application of pretreated and activated mesenchymal stem cells in treatment of myeloid leukemia

Technical Field

The invention belongs to the field of stem cell treatment, and particularly relates to a treatment and activation means of mesenchymal stem cells and a corresponding application of the treatment and activation means in treatment of myeloid leukemia.

Background

Myeloid leukemia is a heterogeneous hematological malignancy, which is mainly characterized by the accumulation of malignant primitive and naive myeloid cells in bone marrow and blood, which stagnate in early stages of differentiation and proliferate clonally in large amounts, seriously hampering ongoing Hematopoietic and immune functions, myeloid leukemia is divided into chronic myeloid leukemia (CM L) and acute myeloid leukemia (AM L), CM L is slow in onset, its natural course includes asymptomatic, chronic, accelerated and acute stages CM L usually starts from chronic stage and progresses to accelerated stage over the course of several years, finally progresses to acute stage, CM L is terminal stage, the clinical manifestations of AM L, the main means for treating AM L is chemotherapy Hematopoietic transplantation (hematotic transplantation CAR), which is still a clinically effective therapy against hsc T-cell transplantation), which is still a major therapy for the clinical adjuvant therapy of CD cells, which is still limited by the clinical T-cell therapy, which is not only a clinically successful therapy for the acute myeloid leukemia, but also has been used for the clinical adjuvant therapy of lymphoblastic leukemia, which is not only for the clinical T-cell transplantation, which is a major cause of the clinical remission of lymphoblastic leukemia, which is a clinical outcome of the lack of the clinical outcome of the acute myeloid leukemia, the clinical leukemia, the lack of the clinical outcome of the clinical leukemia.

The most common clinical Mesenchymal stem cells (Bonemarrow derived MSCs, BM-MSCs) are mainly autologous transplantation and can be used for treating various chronic diseases, but researches in acute leukemia patients show that the BM-MSCs of AM L patients lose vigorous self-renewal capacity and maintenance effect on the growth and function of normal hematopoietic stem cells, and support the growth and proliferation of AM L cells, in addition, researches report that BM-MSCs derived from AM L patients can also protect AM L cells from the effect of chemotherapy drugs, therefore, the autologous transplantation BM-MSCs cannot achieve the effect of relieving the disease course of AM L and even can aggravate the disease course of AM L.

Umbilical cord mesenchymal stem cells (UC-MSCs) are mesenchymal stem cells isolated from Umbilical cord tissue of a newborn. Compared with other adult mesenchymal stem cells, the UC-MSCs have some unique advantages, such as rapid growth, strong cell secretion capacity, lower immunogenicity and the like. Evidence suggests that allogeneic UC-MSCs from healthy newborns have greater immunomodulatory activity and have been successfully used to treat a variety of autoimmune diseases. Meanwhile, some reports indicate that UC-MSCs have certain antitumor activity.

Differentiation-promoting small-molecule drugs represented by all-trans retinoic acid and arsenic compounds have been proved to be capable of promoting acute promyelocytic leukemia (AP L) to cross differentiation disorders, promote differentiation to mature granulocytes, further inhibit proliferation of leukemia cells and reduce malignancy degree.

1, 2-dihydroxy Vitamin D3(1, 25-Dihydroxyvitamin D3, 1, 25D3), also known as Calcitriol (CAS number: 32222-06-3), is the final active form of Vitamin D3 in vivo and is also a Vitamin necessary for maintaining calcium and phosphorus balance in vivo.1, 25D3, by binding to its nuclear receptor Vitamin D Receptor (VDR), activates gene expression downstream of VDR to achieve various physiological functions such as calcium and phosphorus uptake promotion and bone differentiation.in clinical trials related to leukemia, 1, 25D3 has been used to promote differentiation of AM L cells, although some patients are relieved, it causes significant Hypercalcemia (Hypercalcemia), directly results in clinical trials of 1, 25D3, therefore, optimizing VDR-related therapies becomes a new research to enhance differentiation control of cells AM L, 2-pretreated MSCs, thus enhancing differentiation control of AM 734.

Disclosure of Invention

The invention aims to construct a preparation method of pretreated activated MSCs, and the pretreated activated MSCs are used as a treatment tool for treating myeloid leukemia. The method comprises a technical method for pre-treating and activating the MSCs, a quality control method and a standard of the treated MSCs, and a method and a standard for evaluating the efficiency of promoting the differentiation of the myeloid leukemia.

In order to achieve the aim, the scheme for pretreating and activating the MSCs is to pretreat and activate the MSCs by 1, 25D3 with the concentration of 1-10 mu M so as to enable the MSCs to highly express interleukin 6(I L-6) and indole-2, 3 dioxygenase (IDO), and further to efficiently induce AM L cells to differentiate, thereby establishing a brand-new AM L differentiation therapy.

The specific treatment method comprises the following steps:

1. 1×10⁶the MSCs cells were passaged to 60mm by 15mm dishes using α -MEM complete medium containing 10% FBS at 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator.

2. 1, 25D3 was added to the complete medium to a final concentration of 1-10. mu.M in the culture broth. Under these conditions, MSCs were cultured for 24-48 hours.

3. After treatment with 1, 25D3, the supernatant was discarded, washed twice with PBS, and fresh α -MEM complete medium containing 10% FBS was replaced to continue culturing MSCs for subsequent use (such MSCs are defined herein as Active MSCs).

The quality control method and standard of the Active MSCs are as follows:

this section describes whether or not the pretreated MSCs, i.e., Active MSCs, meet the quality control standards for subsequent application to myeloid leukemia differentiation therapy, and the methods for determining the same.

Quality control of 1, 25D3 residue content in culture supernatant of Active MSCs by detecting 1, 25D3 content in the supernatant with high performance liquid chromatography (HP L C), wherein the 1, 25D3 content in the supernatant should be below 0.1 nM.

Quality control of cell viability of Active MSCs: the CCK8 cell proliferation assay detects the cell proliferation capacity of Active MSCs. The proliferation capacity of Active MSCs should not be significantly altered compared to that before treatment.

Quality control of Active MSCs phenotype: flow cytometry identified the expression of Active MSCs cell surface markers CD14, CD34, CD45, CD73, CD90, CD 105. In contrast to the pretreatment, cells from Active MSCs indicate that the abundance of marker expression should not change significantly.

Active MSCs multipotency evaluation: the Active MSCs are subjected to adipogenic, osteogenic and chondrogenic induced differentiation, the degree of differentiation is evaluated, and the maintenance capacity of the pluripotency is judged. Compared with the Active MSCs before treatment, the adipogenic cell differentiation, the osteoblastic cell differentiation and the chondroblastic cell differentiation capacity of the Active MSCs are not affected.

The Active MSCs meeting the above standards can be applied to the next step of promoting the differentiation of myeloid leukemia cells.

1. The expression of interleukin 6(I L-6) and indole-2, 3 dioxygenase (IDO) of the Active MSCs is measured by using real-time quantitative PCR (RT-qPCR) and E L ISA, compared with the expression and secretion capacity of I L-6 and IDO of the Active MSCs before pretreatment, the expression and secretion capacity of the I L-6 and the IDO of the Active MSCs are improved by more than 3 times.

2.Active MSCs were co-cultured with human leukemia cell lines (THP-1 and U937 cell lines) or primary leukemia cells from leukemia patients, respectively, and the expression of CD14 and CD11b on AM L cells was assessed by flow cytometry, the degree of expression of CD11b, CD14 double positive was up-regulated by at least 50% relative to that before treatment.

3.Active MSCs are respectively cultured with CFSE labeled human leukemia cell lines (THP-1 and U937 cell lines) for 1-3 days, and the proliferation of AM L cells is remarkably inhibited by the Active MSCs detected by flow cytometry.

Compared with untreated MSCs of the same batch, the Active MSCs prepared by the invention have better capacity of inducing the differentiation of leukemia cells, and MSCs activated by using 1, 25D3 pretreatment have stronger secretion capacity, wherein the expression of a key protein I L-6 for inducing the differentiation is obviously increased.

The effective dose of the Active MSCs for treating leukemia by peripheral intravenous infusion is (5-10) × 10⁶In terms of/kg. The effective dose is determined by achieving a therapeutic effect in a number of animal model tests.

Drawings

FIG. 1 high performance liquid chromatography (HP L C) detects 1, 25D3 residues in pretreated MSCs (A) same batch of untreated MSCs, (B) Active MSCs, and (C)1, 25D3 pure product.

FIG. 2 CCK8 examined the effect of 1, 25D3 on the proliferation of MSCs.

FIG. 3 flow assay of Active MSCs detecting expression of markers CD73, CD90, CD105, CD14, CD34, CD 45.

FIG. 4 identification of induced differentiation of osteogenic (A), adipogenic (B) and chondrogenic (C) MSCs

FIG. 5 is a test of the ability of MSCs to induce differentiation of acute monocytic leukemia cells in vitro flow cytometry tests (A) the expression of CD14 and CD11B on the cell surface of the AM L cell line U937 and (B) the expression of CD14 and CD11B on the cell surface of primary samples of leukemia patients.

FIG. 6 measurement of the ability of MSCs to inhibit the proliferation of acute monocytic leukemia cells in vitro. CCK8 was tested for proliferation of THP-1 (left) and U937 (right) cell lines.

FIG. 7 expression levels of activated MSCs, I L-6 and IDO were pretreated.

FIG. 8 detection of leukemia inhibitory effect of pretreated activated MSCs on mice following intravenous infusion.

Detailed Description

The principle features and technical solutions of the present invention are described below with reference to examples, which are only used for explaining the present invention and are not used for limiting the scope of the present invention.

Example 1

Separation and subculture of umbilical cord mesenchymal stem cells

1. Preparing tissue digestive enzyme (type II collagenase 250U/ml, neutral protease 100U/ml, hyaluronidase 10U/ml), dissolving in α -MEM culture medium at 37 deg.C, filtering with 0.22 μm filter, and sterilizing;

2. collecting umbilical cord tissue of newborn infant, cleaning umbilical cord in culture dish with PBS containing 0.1% penicillin-streptomycin double antibody, placing in α -MEM culture medium, peeling off three blood vessels, and cutting umbilical cord into pieces of 1-2mm³The tissue mass of (a);

3. mixing the cut tissue blocks and the prepared tissue digestive enzyme solution in a volume ratio of 1: 1 in a 50ml centrifuge tube, digesting for 3 hours at 37 ℃ and 200rpm until the tissue blocks are basically completely digested;

4. centrifuging the digested tissue fluid at 4 deg.C and 300g for 5min, discarding supernatant, resuspending PBS in 50ml α -MEM medium, centrifuging at 4 deg.C and 300g for 5min, discarding supernatant, washing with PBS twice, resuspending the precipitate in α -MEM medium containing 10% fetal calf serum and 1% double antibody, inoculating in 10cm diameter cell culture dish, standing at 37 deg.C and 5% CO₂Standing in a saturated humidity incubator for adherent culture;

after 5.3 days, half the liquid change. Thereafter, every two days, fluid is changed, MSCS outgrow along adherent tissue blocks or adherent cells;

6. when the cells grow to 80% abundance, digesting the lower cells by using cell digestive juice containing 0.25% of pancreatin and 0.02% of EDTA; resuspending the cells, centrifuging at 1000rpm for 5min, discarding the supernatant, washing with PBS, centrifuging, discarding the supernatant, resuspending the obtained cell pellet with fresh culture medium, inoculating to a new culture dish, and passaging the cells. After the cells grow to 90% confluence, the next subculture is performed.

Example 2

Optimization of preparation method of pretreated activated mesenchymal stem cells

1. As a preferred embodiment of the pre-treatment activation of the MSCs of the invention, the final concentration of 1, 25D3 in the culture broth is 4. mu.M.

2. As a preferred embodiment of the pre-treatment activation of the MSCs of the present invention, the MSCs are cultured to an abundance of 70-80%.

3. As a preferred embodiment of the present invention for the pre-treatment activation of MSCs, MSCs are added to 1, 25D3 and cultured for 24 hours.

4. As a preferred embodiment of the present invention, MSCs are activated by pretreatment for 24 hours, the supernatant is discarded, washed twice with PBS (pH7.4), and cultured in α -MEM medium containing 10% FBS.

Example 3

The quality control method and standard of the Active MSCs are as follows:

quality control of 1, 25D3 residual content in culture supernatant of Active MSCs comprises washing Active MSCs twice with PBS, culturing in α -MEM complete culture medium containing 10% FBS at 37 deg.C and 5% CO2 incubator for 2 days, collecting culture supernatant, and detecting 1, 25D3 content in the supernatant by high performance liquid chromatography (HP L C), wherein the content of 1, 25D3 in the supernatant should be below 0.1 nM.

Quality control of cell viability of Active MSCs: CFSE tested the cell proliferation capacity of Active MSCs as a control of untreated MSCs from the same batch. The proliferative capacity should be unchanged compared to before treatment.

Quality control of Active MSCs phenotype: the expression of the Active MSCs cell surface markers CD14, CD34, CD45, CD73, CD90, CD105 was identified by flow cytometry as controls on untreated MSCs from the same batch. This indicates that marker expression should not be significantly altered compared to before treatment.

Active MSCs multipotency evaluation: using untreated MSCs of the same batch as a control, and simultaneously carrying out adipogenic, osteogenic and chondrogenic induced differentiation and identification on Active MSCs: adopting MSCs osteogenic and adipogenic differentiation induction culture medium and induction scheme of Guangzhou Seisakuchen organisms to induce adipogenic and osteogenic differentiation; inducing chondrogenic differentiation by adopting MSCs chondrogenic differentiation induction culture medium and induction scheme of Stem Cell. The strength change of the differentiation capacity is judged by morphological staining and observation. The differentiation capacity of the three lineages should not change significantly compared to that before pretreatment.

Example 4

The evaluation method and the standard of the efficiency of the Active MSCs for promoting the AM L cell differentiation are as follows:

active MSCs, and untreated MSCs of the same batch were each treated at 10⁵Inoculating to 12-well plate, culturing in incubator for 12 hr, discarding culture supernatant, washing with PBS for 2 times, and directly inoculating 2 × 10 with human leukemia cells (THP-1, U937 cell line, primary leukemia cells) cultured under the same culture conditions⁵THP-1 and U937 cells/well in plates cultured for MSCs and Active MSCs, in vitro experiments were performed.

1. After 48h of co-culture, the separately cultured or co-cultured THP-1 and U937 cell lines were harvested into 1.5ml EP tubes, washed twice with PBS, and the co-cultured THP-1 and U937 cell lines were examined for expression of CD14 and CD11b and changes in phagocytic capacity by flow cytometry using the separately cultured THP-1 and U937 cell lines as controls.

2. The expression of interleukin 6(I L-6) and indole-2, 3 dioxygenase (IDO) in Active MSCs is detected by real-time quantitative PCR (RT-qPCR) by using untreated MSCs of the same batch as a control, and is increased by 3 times or more.

The foregoing examples further illustrate the present invention but are not to be construed as limiting thereof. It will be apparent to those skilled in the art that modifications and substitutions to methods, steps or conditions of the invention can be made without departing from the spirit and substance of the invention.

Claims

1.A preparation method of activated mesenchymal stem cells comprises the following steps:

1) the MSCs cells were passaged to 60mm by 15mm dishes using α -MEM complete medium containing 10% FBS at 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator to achieve a density of 80%.

2) 1, 25D3 was added to the complete medium to a final concentration of 1-10. mu.M in the culture broth. Under these conditions, MSCs were cultured and activated for 24 hours.

3) After one day of treatment with 1, 25D3, the supernatant was discarded, washed twice with PBS, and fresh α -MEM complete medium containing 10% FBS was replaced to continue culturing MSCs for subsequent use (such MSCs are defined herein as Active MSCs).

2. An activated mesenchymal stem cell according to claim 1, characterised in that it uses mesenchymal stem cells of xenogenic origin, such as: umbilical cord tissue-derived mesenchymal stem cells (UC-MSCs) of neonates.

3. The method of claims 1 and 2, wherein the small chemical molecule (e.g. 1, 25D3) is activated to activate Vitamin D Receptor (VDR) of mesenchymal stem cells, so that interleukin 6(I L-6) and indole-2, 3 dioxygenase (IDO) are highly expressed, thereby inducing differentiation of acute monocytic leukemia cells.

4. The method for preparing activated mesenchymal stem cells according to claim 3, wherein the stimulation time of the umbilical cord mesenchymal stem cells by the 1, 25D3 is 24-48 h.

5. The 1, 25D3 activated mesenchymal stem cell according to claim 4, wherein the 1, 25D3 method for activating umbilical cord mesenchymal stem cells comprises: the final concentration of 1-10. mu.M 1, 25D3 stimulated mesenchymal stem cells.

6. The activated mesenchymal stem cell of claim 5, wherein the small chemical molecule (VDR agonist 1, 25D3 and its modified product such as delphirin) activates VDR in the mesenchymal stem cell to increase the expression level of I L-6 and IDO expressed by MSC by more than 3 times, and accordingly, the differentiation degree of leukemia cell (including cell line and primary cell) should be adjusted by at least 50% (the differentiation degree is determined by the double positive expression of CD11b and CD 14).

7. The use of a pretreated activated mesenchymal stem cell according to claim 5 as a novel differentiation therapy of myeloid leukemia (acute myeloid leukemia and chronic myeloid leukemia).

8. According toThe mesenchymal stem cell of claim 6, wherein the effective amount for peripheral intravenous infusion in the treatment of AM L is 5 × 10⁶/kg-10×10⁶/kg。