三种间充质干细胞向神经元样细胞的诱导与分化

doi:10.3969/j.issn.2095-4344.0671

摘要/Abstract

摘要：

文章快速阅读：

文题释义：
共培养：是为了建立更类似于体内环境的培养体系，尽可能使体外环境与体内环境相吻合，从而使细胞间能相互沟通信息，相互支撑生长增殖而在细胞培养技术的基础上发展出的培养技术。细胞共培养技术是将2种或2种以上的细胞共同培养于同一环境中，由于其具有更好地反映体内环境的优点，被广泛应用于现代细胞研究中。
Transwell小室：是一种以膜滤器隔离培养的体系。以其进行共培养时，常采用小于3.0 μm孔径，以防止细胞发生迁徙。可将细胞A种于上室，细胞B种于下室，可以研究细胞B分泌或代谢产生的物质对细胞A的影响。

摘要
背景：作为采集方便、来源广泛、潜能巨大的细胞来源，干细胞在疾病细胞疗法的研究已发展壮大，其中最常见使用的干细胞有骨髓间充质干细胞、胎盘间充质干细胞和脐血间充质干细胞。
目的：比较骨髓间充质干细胞、胎盘间充质干细胞和脐血间充质干细胞与神经细胞的共培养后的变化。
方法：体外分离培养人骨髓间充质干细胞、人胎盘间充质干细胞和人脐血间充质干细胞，通过Transwell平板与神经细胞共培养，观察间充质干细胞分化成神经细胞的形态及神经元特异性烯醇化酶表达的变化。
结果与结论：神经共培养系统中3种间充质干细胞均逐渐变为星状，并出现相互连接，且阳性表达神经元特异性烯醇酶。表明神经细胞提供的微环境可以诱导及促进3种间充质干细胞向神经元样细胞分化。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0002-8137-2054(Yang Ying)

关键词: 骨髓间充质干细胞, 人胎盘间充质干细胞, 脐带血液间充质干细胞, 细胞分化, 神经细胞, 共培养, 干细胞

Abstract:

BACKGROUND: Stem cells, characterized with convenient collection, wide sources and great potential, have been developed in the cell therapy. The most commonly used stem cells include bone marrow mesenchymal stem cells, placental mesenchymal stem cells and umbilical cord blood mesenchymal stem cells.
OBJECTIVE: To compare the biological changes in bone marrow mesenchymal stem cells, placental mesenchymal stem cells and umbilical cord blood mesenchymal stem cells after co-cultured with nerve cells.
METHODS: Human bone marrow mesenchymal stem cells, human placental mesenchymal stem cells and human umbilical cord blood mesenchymal stem cells were isolated and cultured in vitro. The morphological changes of mesenchymal stem cells differentiated into nerve cells were observed by co-culture with Transwell culture plates. The expression of neuron-specific enolase was detected after induction.
RESULTS AND CONCLUSION: In the co-culture system, these three kinds of mesenchymal stem cells gradually formed a star-like shape and were interconnected. Moreover, all the cells were positive for neuron-specific enolase. These findings reveal that the microenvironment provided by nerve cells can induce and promote the differentiation of three kinds of mesenchymal stem cells into neuron-like cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Mesenchymal Stem Cells, Coculture Techniques, Tissue Engineering

中图分类号:

R394.2

杨颖，徐倩，王会玲，姚民秀. 三种间充质干细胞向神经元样细胞的诱导与分化[J]. 中国组织工程研究, 2018, 22(33): 5356-5361.

Yang Ying, Xu Qian, Wang Hui-ling, Yao Min-xiu. Three kinds of mesenchymal stem cells: differentiating into neuron-like cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(33): 5356-5361.

图/表（结果） 2

参考文献

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引言

Bone marrow mesenchymal stem cells (BMSCs) are the early cells of mesoderm development^[1], and have self-renewal and multi differentiation capacities, which have been widely used in the regenerative medicine^[2-4]. The latest studies have shown that mesenchymal stem cells (MSCs) can survive, proliferate, migrate and differentiate into neuron-like cells in the damaged brain tissue and spinal cord^[5-8]. Sanchez-Ramos et al.^[9] established a culture system in which neurotrophic factor V was mainly used to induce MSCs differentiation into neurons in vitro. Woodbury et al.^[10-11] found that adult rat and human MSCs can differentiate into neurons under induction by specific factors. Kopen et al.^[12] injected MSCs into the lateral ventricles of newborn rats, and found that the MSCs were differentiated into neurons and glial cells, with a certain ability of migration, indicating that MSCs have more extensive differentiation potential. However, it has not been reported that whether nerve cells can directly induce MSCs to differentiate into neurons.

Human placental MSCs (hPMSCs) have the ability to differentiate into multiple embryonic germ cells from various tissues^[13-19]. Zhang et al.^[20] transplanted MSCs that marked by bromodeoxyuridine via the carotid artery, jugular vein, and lateral ventricle. Part of cells migrated to the hippocampus differentiated into neurons, and MSCs migrated into the cortex differentiated into oligodendrocytes. Nailong et al.^[21] co-cultured newborn rat’s brain cells with hPMSCs, and the hPMSCs then differentiated into neuron-like cells^[22]. How to place MSCs in a microenvironment close to the human body thereby inducing the differentiation into nerve cells or improving the nutrition and metabolism of nerve cells to repair damage to the nervous system has become the recent research focus of cell replacement therapy in clinical practice.

Compared with stem cells from other sources, umbilical cord blood MSCs (UC-MSCs) have the advantages of weak immunogenicity, wide source, convenient collection, more primitive, more immature, stronger ability of proliferation, and no ethical issues^[23]. As a cell source of cell therapy for many diseases, UC-MSCs have a broad prospect in clinical application^[24].

In view of this, Transwell co-culture system was used in this study to in vitro co-culture hBMSCs, hPMSCs, UC-MSCs with nerve cells, aiming to find out whether MSCs can be induced to differentiate into neurons in the microenvironment provided by nerve cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

材料方法

Design

A cytology experiment.

Time and setting

The study was completed at the Central Laboratory, Second Affiliated Hospital of Qingdao University Medical School in China from January 2016 to September 2016.

Materials

Bone marrow samples were isolated from diabetic patients who were treated by autologous stem cell transplantation. Nerve cells were harvested from the brain tissue of asphyxiated newborn infants who died during the delivery process. Placental samples were provided by the Department of Obstetrics and Gynecology in Qingdao Municipal Hospital, China. Specimen collection standards are as follows: human full-term cesarean section fetus, no maternal infectious disease, no abnormal fetus. Umbilical cord blood samples were provided by the Department of Obstetrics and Gynecology in Qingdao Municipal Hospital. Specimen collection standards are as follows: full-term normal delivery, no infectious diseases and no family genetic diseases.

All the specimens were strictly collected under sterile operation. Informed consent was obtained from each patient or his/her legal representatives, as well as the permission from the material provider. This experiment was approved by the Hospital Ethics Committee of Qingdao Municipal Hospital (approval No. NCT03098771).

Nestin antibody was purchased from Boster, Wuhan, China; fluorescent secondary antibody was purchased from Sigma (St. Louis, MO, USA); low sugar DMEM medium purchased from Gibco, Steuben County, NY, USA; fetal bovine serum (FBS) purchased from Hangzhou Sijiqing Biological Engineering Company, China; basic fibroblast growth factor (bFGF) purchased from Sigma; neuron-specific enolase (NSE) antibody purchased from Boster; Transwell system purchased from Corning Costar (Steuben County, NY, USA).

Methods

Nerve cell culture method

After rinsing with D-Hank’s solution 2-3 times, the cerebral cortex were cut into pieces, pipetted, mixed completely with the culture medium, and filtered using 40 μm filter mesh. The suspension was adjusted to the cell density of 5×10⁵-10×10⁵ cells/L, followed by vaccination to the culture bottle. Twenty-four hours later, the growth of adherent cells was observed. The culture medium was semi-changed at 2-3 days, and passaged at 5-7 days.

Culturing method for MSCs

Under inverted optical microscope, the cell growth and proliferation were observed, and passage 3 BMSCs, hPMSCs, and UC-MSCs were collected using Percoll gradient centrifugation (relative density 1.007). Mononuclear cells from the interface layer were centrifuged at 1 000 r/min for 10 minutes, followed by the pre-cooling, and washing in pH 7.0 PBS twice, 2.5% glutaraldehyde fixation 4 ^oC overnight. JEM2100CX was used to observe the cell morphology. The collected mononuclear cells were incubated at room temperature with single antibody labeled CD34 and CD44 for 15 minutes. The percentage of CD34⁺ and CD44⁺ cells (lymphocytes and monocytes) in the mononuclear cells was determined using flow cytometry eliesp (COULTER) within 6 hours^[7,10,25].

Co-culture

Passage 3 nerve cells were inoculated on the bottom of the Transwell system with the density of 1×10⁶/mL. At the same time, the Transwell’s insert was placed into the 6-well plate. After MSCs inoculation, the culture medium in the insert and the 6-well plate could exchange and mix together. Thus, we set up the co-culture system. And in the control group, we inoculated MSCs on both the bottom of the Transwell system and the insert. Morphological changes of cells and the expression of NSE were observed under the inverted microscope^[5].

Detection of NSE positive cells

Cell samples were fixed with 4% paraformaldehyde at room temperature for 1 hours, washed in PBS thrice, mounted with 0.3% H₂O₂ for 30 minutes at room temperature, washed in PBS thrice, mounted with goat serum at 37 ^oC for 30 minutes, incubated with NSE resistance (1: 100 dilution) at 37 ^oC for 1.5 hours, washed in PBS thrice, labeled with biotin at 37 ^oC for 30 minutes, rinsed in PBS thrice, labeled with horseradish peroxidase at 37 ^oC for 30 minutes, rinsed in PBS thrice, developed with DAB chromogenic. Then, PBS was used to wash off the floating color. We randomly selected eight non-overlapping fields of vision (100×) to calculate the ratio of NSE positive cells to the total number per field of vision.

Main outcome measures

The morphological changes and NSE expression of positive cells were investigated.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

讨论

CD34 is a lipopolysaccharide receptor positive for hematopoietic stem/progenitor cells and endothelial cells, and CD29 and CD44 are the specific surface markers of stromal cells. It is generally believed that positive expression of CD44 (a cell surface adhesion molecule) is a specific sign of MSCs. As MSCs do not express hematopoietic stem cell surface antigens, such as CD34 (a hematopoietic precursor cell marker antigen), immunofluorescence detection of CD44 and CD34 can be used as one of the methods for the identification of MSCs. In this study, passage 3 cells showed positive expression of CD44 and negative expression of CD34^[25].

In clinic, BMSCs transplantation into the patient body has been succeeded, and has been used for the treatment of various diseases, such as bone tissue defect and myocardial infarction^[26-27]. BMSCs will have extensive clinical treatment in the future.

MSCs are derived from the early development of mesoderm and ectoderm, and the main part of the placenta is amniotic and chorionic mesoderm, which has the possibility of placenta containing MSCs^[28-32]. hPMSCs also play an important role in the clinical application^[33-34], which can be used for the treatment of neurological diseases, myocardial injury, burns and other diseases^[35-37].

Less is reported on neuronal differentiation of UC-MSCs neurons. With the rise of tissue engineering and transplantation in clinical medicine, UC-MSCs, as the seed cells, with simple separation, low immune original, no ethics and many other advantages have become the hot spot in the stem cell research.

NSE is a kind of acid soluble protein isolated from the brain tissue. As the specificity recognition antibody of neurons and neuroendocrine cells, NSE is commonly used to detect whether neurons exist after the induction and differentiation of stem cells and to study the neuronal development and growth. The higher NSE positive expression rate indicates the better induction^[38]. Therefore, our study used the NSE as the indicator to detect the nerve cells.

Co-culture induction mechanism uses the contact between cells, creates the microenvironment of the nervous system. Cytokines under the system can exchange information and support each other, so as to promote MSCs to differentiate into neurons. MSCs secrete nutrition factors and further promote the growth of nerve cells. The mechanism of co-culture enables MSCs to be placed in a microenvironment that is closer to the human body, inducing its differentiation into nerve cells, which can improve the nutrition and metabolism of the nerve cells, so that the nervous system damage can be repaired^[39]. Our experiments improved the traditional method based on nerve cell separation culture, gently pipetted cell clones, partially decentralized them, as far as possible to reduce the mechanical damage, in order to maintain the original linkage between cells. The results showed that cell death was significantly reduced, clone spheres formed faster, and the microenvironment created was more close to the nervous system of the body.

This experiment attempts to establish the co-culture system by Transwell device. This device is a new type of double layer culture dish, there are interconnected micropores between two layers, and the diameter of the micropore is 3 µm. In this culture system, cells cannot go through the micropore, but some secretory components of the cells can. Therefore, we can co-culture the MSCs with nerve cells, and neural stem cells may have induced material to provide a certain microenvironment to induce differentiation of MSCs.

We found that the MSCs in the co-culture system gradually changed in shape. The cells became stellate, and after 4-5 days of co-culture, stellate cells gradually increased in number and formed interconnection with each other, with neuron-like morphology. Immunofluorescence results showed the cells were positive for NSE. While in the control group, pure MSCs cultured had no a neuron-like morphology, and did no expressed NSE. Therefore, we think that in the process of nerve cell growth, they can secrete some substances to induce MSCs differentiation into nerve cells, which can provide the certain theoretical support for the use of MSCs as the adjuvant treatment to treat nerve system diseases.