诱导剂共培养：谁更适宜骨髓间充质干细胞向神经细胞的分化？

doi:10.3969/j.issn.2095-4344.2013.32.003

中国组织工程研究 ›› 2013, Vol. 17 ›› Issue (32): 5757-5764.doi: 10.3969/j.issn.2095-4344.2013.32.003

• 骨髓干细胞 bone marrow stem cells • 上一篇下一篇

诱导剂共培养：谁更适宜骨髓间充质干细胞向神经细胞的分化？

陈增生¹，褚强²，刘艳凤³，宋璇⁴，李萍⁵

青岛市立医院，¹检验科，²超声科，⁴输血科，山东省青岛市 266071；³青岛市四方区医院检验科，山东省青岛市 266033；⁵青岛大学医学院附属医院输血科，山东省青岛市 266000

收稿日期:2013-05-11 修回日期:2013-06-22 出版日期:2013-08-06 发布日期:2013-08-06
通讯作者: 李萍，硕士，主管技师，青岛大学医学院附属医院输血科，山东省青岛市 266000
作者简介:陈增生★，男，1981年生，山东省招远市人，汉族，2011年青岛大学医学院毕业，硕士，技师，主要从事干细胞方面的研究。

Induction ways of bone marrow mesenchymal stem cells differentiating into nerve cells

Chen Zeng-sheng¹, Chu Qiang², Liu Yan-feng³, Song Xuan⁴, Li Ping⁵

1 Department of Laboratory, Qingdao Municipal Hospital, Qingdao 266071, Shandong Province, China
2 Department of Ultrasound, Qingdao Municipal Hospital, Qingdao 266071, Shandong Province, China
3 Department of Laboratory, Sifang District Hospital, Qingdao 266033, Shandong Province, China
4 Department of Blood Transfusion, Qingdao Municipal Hospital, Qingdao 266071, Shandong Province, China
5 Department of Blood Transfusion, Affiliated Hospital, Medical School of Qingdao University, Qingdao 266000, Shandong Province, China

Received:2013-05-11 Revised:2013-06-22 Online:2013-08-06 Published:2013-08-06
Contact: Li Ping, Master, Technician-in-charge, Department of Blood Transfusion, Affiliated Hospital, Medical School of Qingdao University, Qingdao 266000, Shandong Province, China lpxkck@126.com
About author:Chen Zeng-sheng★, Master, Technician, Department of Laboratory, Qingdao Municipal Hospital, Qingdao 266071, Shandong Province, China Redapple_02@126.com

摘要/Abstract

摘要：

背景：目前骨髓间充质干细胞向神经细胞分化的方法较多，采用不同诱导方法对骨髓充质干细胞分化成神经细胞的比例是不同的。
目的：比较化学诱导法和共培养法诱导大鼠骨髓间充质干细胞分化为神经细胞的差异。
方法：大鼠全骨髓血细胞分离纯化法培养骨髓间充质干细胞，分为化学诱导组和共培养组，分别采用加入化学诱导剂β-巯基乙醇和Transwell小室共培养方法诱导骨髓间充质干细胞向神经细胞分化。
结果与结论：诱导培养1周后从化学诱导和共培养组的骨髓间充质干细胞出现突起，且呈辐射生长，2周后均可见神经元特异性烯醇化酶阳性细胞。共培养组中第四五天可见星级神经细胞状结构，并形成更多的突起，神经元特异性烯醇化酶染色阳性率为(70.82±2.46)%。而在第六七天化学诱导组中神经细胞形态样细胞形成，并有连接，神经元特异性烯醇化酶染色阳性率为(52.37±1.83)%。提示细胞微环境在骨髓间充质干细胞分化成神经细胞发挥主导作用，且化学诱导法诱导效率低于共培养法。

关键词: 干细胞, 骨髓干细胞, 骨髓间充质干细胞, 神经细胞, 诱导分化, 化学诱导法, 共培养法, 神经元特异性烯醇化酶, 干细胞图片文章

Abstract:

BACKGROUND: Currently, bone marrow mesenchymal stem cells can differentiate into nerve cells via many approaches. Different methods for inducing bone marrow mesenchymal stem cells differentiating into nerve cells have different ratios.
OBJECTIVE: To investigate the difference between chemical method and co-culture method to induce the differentiation of rat bone marrow mesenchymal stem cells into nerve cells.
METHODS: Rat bone marrow mesenchymal stem cells were isolated and purified using whole bone marrow culture method, and then randomly divided into two groups: chemical group, β-mercaptoethanol was added; co-culture group, co-cultured in a Transwell chamber.
RESULTS AND CONCLUSION: Visible protrusions from induced cells showed radiation growth at 1 week of induced culture, and neuron-specific enolase staining was positive at 2 weeks of culture. Star-like structure of nerve cells was visible in the co-culture group within 4-5 days of culture, and then more protrusions formed. Meanwhile, the positive rate of neuron-specific enolase was (70.82±2.46)%. After 6-7 days of culture, neuron-like cells formed and were interconnected in the chemical group; while, the positive rate of neuron-specific enolase was (52.37±1.83)%. These findings suggest that cell microenvironment plays a leading role in the differentiation of bone marrow mesenchymal stem cells into nerve cells, and chemical induction method is inferior to the co-culture method.

Key words: stem cells, bone marrow stem cells, bone marrow mesenchymal stem cells, nerve cells, induced differentiation, chemical induction, co-culture, neuron-specific enolase, stem cell photographs-containing paper

中图分类号:

R394.2

陈增生，褚强，刘艳凤，宋璇，李萍. 诱导剂共培养：谁更适宜骨髓间充质干细胞向神经细胞的分化？[J]. 中国组织工程研究, 2013, 17(32): 5757-5764.

Chen Zeng-sheng, Chu Qiang, Liu Yan-feng, Song Xuan, Li Ping. Induction ways of bone marrow mesenchymal stem cells differentiating into nerve cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(32): 5757-5764.

图/表（结果） 4

Morphology of cultured bone marrow mesenchymal stem cells
Lots of erythrocytes covered the entire bottom of culture flask with whole bone marrow culture after inoculation. A large number of blood cells were removed after low glucose Dulbecco’s modified Eagle’s medium was changed at the first time, scattered adherent cells could be seen in the bottom of the bottle, with fusiform or polygonal appearance, and uneven part was a whirlpool-like growth. The formation of multiple fibroblast-like cell clusters could be seen after 6 or 8 days, few round cells could be seen around the cell clusters. Fibroblast-like growth was pooled into a piece after 12 or 16 days. Adherent cells were long spindle-shaped after passage for 2-3 days. Primary cultured bone marrow mesenchymal stem cells had a multiple differentiation potential and maintained a higher purity over 90% after repeated passages^{[22-23, 25-26]} (Figure 1).

Surface marker expression of bone marrow mesenchymal stem cells
Flow cytometry results showed that bone marrow mesenchymal stem cells strongly expressed hyaluronan receptor CD44, with the positive rate of over 99%, but did not express the hematopoietic stem cell marker CD34 (< 1%) (Figure 2).

Co-culture and chemical induction methods for inducing differentiation into nerve cells

Most of the cultured nerve cells grew adherent, and the cell body was full and fusiform, withshining brilliantly cytoplasm. Small protrusions were visible in the cell body for 2 or 3 days. Glial cell body was flat and had slightly larger size, and one or four short rod protrusions were around the cell body. The nerve cell bodies grew larger, and rounded protrusions around the cell body were longer and increased to form a unipolar, bipolar or multipolar projection for 6 days.

Nerve cells inoculated in the 6-well plates of Transwell co-culture system, bone marrow mesenchymal stem cells were found to have pyknosis smaller than flat cells. Formerly, most of the cell bodies had rounded protrusion formation, and cells gradually became long stellate for 3 days. The stellate cells gradually increased, and could cross each other to form a connection with a neuron-like morphology (Figure 3) for 4 or 5 days. Neural-like cells accounted for 62.4% of the total cells. Immunofluorescence results showed Neuron-specific enolase positive rate was (60.47± 1.89)%, suggesting the performance characteristics of neurons (Figure 3). Most of the cells were still flat and wide long spindle-shaped similar as morphology characteristics of bone marrow mesenchymal stem cells, and did not form a neuron-like morphology in the control group after cultured for 8 days. Neuron-specific enolase was also negative in the control group.

Chemically induced culture results
Cells showed adherent growth after 1-3 days in the chemically induced group, and no obvious morphological changes were observed. The cell body significantly shrank and a circle or a triangle extended at 4 days. Some cells formed longer dendritic-like branches, and mutual extending connection; some cells were similar to neurons, and the long axons appeared to render the nerve cell-like morphology (Figure 4) at 5-6 days. Nerve cells accounted for 50.4% of the total cells, and the positive rate for neuron-specific enolase was (40.91± 1.63)%.

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引言

Stem cells have a capacity of unlimited or longer-term self-renewal and can produce highly differentiated cells at least with multiple differentiation potential and self-replication. Mesenchymal stem cells can be differentiated into different functional cells and form a variety of tissues and organs under certain conditions, such as inducers or differentiation technology. Seed cells for cell transplantation are an important worldwide research direction in the field of tissue engineering. Especially, bone marrow mesenchymal stem cells derived during the development of the early mesoderm can be differentiated to different types of cells in specific conditions such as bone, cartilage, skeletal muscle, tendon and fat cells. Meshenchymal stem cells have advantages of convenient collection, easy culture in vitro, amplification and induction characteristics. Now, it has already be proved that mesenchymal stem cells exist in the bone marrow, liver, spleen, muscle, brain, skin and other tissues. Bone marrow is the main source of mesenchymal stem cells. Bone marrow mesenchymal stem cells are the early cells of mesoderm development^[1], discovered as multi-potential stem cells existing in the marrow cavity, which have unique immunological characteristics, self-renewal

and differentiation capabilities and have been widely used in regenerative medicine. Bone marrow mesenchymal stem cells can differentiate into nerve cells, osteoblasts and fat cells in vivo and in vitro. Nerve cells transplantation brings a hope for the treatment of many central nervous system diseases.

Bone marrow mesenchymal stem cells have superiority in wide sources, which are easily obtained and have strong proliferation. In recent years, mesenchymal stem cells can be induced to differentiate into specific neurons, such as cholinergic neurons, dopaminergic neurons, amino acid neurons, peptidergic neurons^[2]. Recent studies have shown that bone marrow mesenchymal stem cells can survive in the damaged brain tissue and spinal cord, proliferate, migrate, and differentiate into neuron-like cells, thereby improving the neurological function and survival status of patients following spinal cord injuries, stroke and other nervous system diseases effectively^[3-4].

Neurotrophic factors play a positive role in the repair process of the central nervous system, which can restore spinal cord function. Neurotrophic factor and brain-derived neurotrophic factor are particularly important. Neurotrophic factor is the first importmant factor for regulating cell growth which has dual biological functions: neurons nutrition and promotion of neurite outgrowth. Brain-derived neurotrophic factor plays an important role in the development and differentiation of neurons and its renewable growth. Woodbury et al ^[5-8]co-cultured the nestin-positive cells from the cord blood with primary cells from rat cerebral cortex for 4 days, 40% of which differentiated into neurons, including 30% astrocytes and 11% oligodendrocytes. Such conditions are closer to the physiological state of nerve cells during the growth process.

There are two common methods used for induced differentiation, one is chemical induction method which increases intracellular cyclic adenosine monophosphate by antioxidant induction and growth factors^[9-11]; the other is co-culture method which use intracellular microenvironment of mesenchymal stem cells^[12-20]. By the comparison of two induction methods in the experiment, we aimed to find a more simple, economical and practical way for induced differentiation into nerve cells.

材料方法

Design

Comparative cell culture study.

Time and setting

The experiment was completed from September 2011 to October 2012 in Qingdao University Medical Laboratory.

Materials

Animals

Clean healthy adult male Sprague-Dawley rats, aged 6 to 8 weeks, weighing 280-320 g, were provided by Shandong Experimental Animal Center, license No. SCXK (Lu) 20050016. The experimental procedures on animals were consistent with standards of medical ethics.

Main reagents for induced differentiation are as follows:

Experimental methods

Primary culture of bone marrow mesenchymal stem cells

After rats were sacrificed, 2 mL femur bone marrow blood were extracted and added into 10 mL low glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum under sterile conditions, winded and mixed with a straw gently, and inoculated in a sterile culture bottle directly that was placed into a 37 ℃ incubator with 5% CO₂. Dulbecco’s modified Eagle’s medium was at the first time changed after 48 hours, and then changed every 3 or 4 days. The cells were digested and passaged when grew to cover 85% of the space with 0.25% trypsin at room temperature. The obtained nucleated cells were counted and surface cell markers were identified after the third passage^[21-22].

Identified cell surface markers of bone marrow mesenchymal stem cells

Passage 3 bone marrow mesenchymal stem cells were trypsinized at cell density of 1.0×10⁹/L. Mouse anti-human antibody CD44 and CD34 were added to the centrifuge tube containing 0.2 mL bone marrow mesenchymal stem cells by immunofluorescence. The control group was added with 0.04 mL Fitc and 0.02 mL PE, at 4 ℃ in the dark reaction for 30 minutes, stored in 0.2 mL paraformaldehyde after 1 500 r/min centrifugation, analyzed by flow cytometry.

Co-culture induced differentiation

Rat brain tissue under sterile conditions was added into low glucose Dulbecco’s modified Eagle’s medium after quickly weighing, on ice for 10 minutes, followed by shear separation, wind and percussion, over 40 μm filter at 4 ℃. The supernatant was collected, adherent cells growth at cell density of 5×10⁵/L could be seen after suspension culture for 24 hours, exhibiting round or irregular-shaped appearance. Half of the medium was changed to remove suspended cells which continued to be cultured in the high glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Then, adherent cells exhibited reticular growth under an inverted microscope. The whole medium was changed every 3 days. Typical nerve cell structure was visible after 7 days^[23].

Nerve cells at a density of 1×10⁶/L were seeded in the bottom of the Transwell bunk, while Transwell dish was inserted to inoculate rat bone marrow mesenchymal stem cells. The culture media in the insert and 6-well plates were low glucose Dulbecco’s modified Eagle’s media that could interpenetrate each other. Common medium was used in the Transwell bunk dish of bone marrow mesenchymal stem cells in the control group for vaccination. Morphological changes of bone marrow mesenchymal stem cells and immunofluorescence detection of neuron-specific enolase expression were observed.

Chemically induced differentiation

Bone marrow mesenchymal stem cells at 1×10⁸/L after the 3^rd generation amplification in vitro were seeded in 6-well plates, disinfectant coverslip per orifice was added to serum-free medium serum substitute of 10% volume fraction, then pre-inductioned in 2 mL low glucose Dulbecco’s modified Eagle’s medium containing 3 mmol/L β-mercaptoethanol for 24 hours until about 60% cells confluent. After washing in PBS twice (pH 7.4), the medium was replaced by 20 g/L dimethyl sulfoxide, 200 mmol/L butylated hydroxy anisole serum-free medium for continuous induced culture. Rat bone marrow mesenchymal stem cells of the control group^[24] were cultured in a pure low glucose Dulbecco’s modified Eagle’s medium.

Nerve cells identified by immunohistochemistry

The induced cells of chemical and co-culture groups were fixed for 1 hour with 4% paraformaldehyde formaldehyde at room temperature, washed with PBS×3, closed with 0.3% hydrogen peroxide for

10 minutes at room temperature, washed with PBS three times, cultured in 37 ℃ goat serum for 15 minutes, incubated with neuron-specific enolase antibody (1:100 dilution) for 1.5 hours at 37 ℃, washed with PBS three times, followed by addition of biotin-labeled secondary antibody at 37 ℃ for 30 minutes, washed with PBS three times, horseradish overperoxidase at 37 ℃ for 45 minutes, washed with PBS three times, 3,3’-diaminobenzidine staining for 4 minutes, the chromogenic hematoxylin was ended with PBS thenobserved under inverted microscope, the cytoplasm of positive cells was stained brown,

10 non-overlapping horizons (×100) were randomly counted to calculate neuron-specific enolase positive cells, respectively, accounting for the view counting the proportion of the total number of cells (positive rate = positive cell number/total number of cells), complex piece. The bone marrow mesenchymal stem cells were stained with neuron-specific enolase immunofluorescence, after oriental induction and fixation, to analyze the percentage of neuron-specific enolase positive cells in each field of view with the use of the Image Pro Plus 6.0 image analysis software. Inducible factor was added into the experimental group, but nothing in the negative control group. Three wells were selected every time and observed at 10-fold vision.

Main outcome measures

Bone marrow mesenchymal stem cells induced using chemical method, including β-mercaptoethanol, dimethyl sulfoxide and butylated hydroxy anisole, were compared to those induced by co-culture method. Neuron-specific enolase was immunohistochemically identified.

Statistical analysis

Results were expressed as mean±SD and analyzed by t-test. A value of P < 0.05 was considered as statistically significant.

讨论

Stem cells are a group of primitive cells, capable of self-renewal. Human bone marrow is the body’s largest stem cell pool, including hematopoietic stem cells, mesenchymal stem cells and endothelial progenitor cells from stem cells. Mesenchymal stem cells^[27-30] are a kind of cells originated in the early mesoderm and ectoderm, have a capacity of infinite or eternal self-renewal, can produce at least one type of the differentiated progeny cells highly. Mesenchymal stem cells have multi-differentiation potential and self-renewal capacity, showing easy separation and amplification, to repair damaged tissue by cell transplantation. Experimental studies in vitro have shown that mesenchymal stem cells are the most promising seed cells for a wide application range, such as tissue engineering. Mesenchymal stem cells can differentiate into bone, fat, muscle, and other sources of mesoderm tissues under certain conditions, become a concern plasticity of adult stem cells as a source of cells to repair these tissues.

With the rise of tissue engineering and clinical transplantation medicine, bone marrow mesenchymal stem cells have biological characteristics and diversity of sources as seed cells. Bone marrow mesenchymal stem cells have capacity of self-renewal, the advantage goes without saying that to avoid the immune rejection and ethical issues inherent in other stem cells, thus making it a promising nerve graft material. The differentiation of bone marrow mesenchymal stem cells into nerve cells in vitro with the following advantages: Bone marrow mesenchymal stem cells are induced to differentiate into specific nerve cells to treat a particular nervous system disease, which should be more targeted and effective. It is generally believed that the high degree of cells differentiation relatively corresponds to small malignant. Thorough understanding differentiation of stem cells under transdifferentiation conditions may help people gain a deeper understanding of the mechanism of the disease. Avoiding the ethical problems of the application of neural stem cells in nerve regeneration, bone marrow mesenchymal stem cells can differentiate into nerve cells and glial cells. Mesenchymal stem cells in the bone marrow can be as artificial neural tissue engineering seed cells. Bone marrow mesenchymal stem cells differentiate into nerve cells in the ability to shape characteristics of self-renewal capability, the ability of the ultimate differentiation of neurons and glial cells, secretion of neurotrophic factors can be observed in a large number of experiments that bone marrow mesenchymal stem cells differentiate into nerve cells. Other aspects have shown the advantages. Adult non-nerve tissue mesenchymal stem cells induced nerve cells have achieved self-renewal and proliferation through the symmetrical split and asymmetric division. Daughter cells are identical to the parent generation with the biological characteristics of stem cells after the split. Neurospheres growth and proliferation shows good self-renewal and differentiation potential.

Chemical induction method for cell proliferation and differentiation can be affected by a variety of cytokines. Chemicals and neurotrophic factors are added to induce mesenchymal stem cells into nerve cells in the chemical induction method, but the mechanism underlying induction of differentiation is unclear. Some studies pointed out that toxic chemical substance may cause a normal cell morphology change making the cell structure shrinkage and producing nerve cell-like morphology in the chemical induction method. Nerve impulses do not produce or have the function of the nerve cells by the induced method. Most of the inducing agents for the chemical method are common antioxidants, so researchers believe that the survival time of nerve cells in vitro can be enhanced^[31-32] through antioxidation. Chemical induction method is flawed mainly in the following aspects: a large amount of cytokines, the higher cost. The growth factors that are used in clinical practice may cause systemic reactions of the body. The inducted cells are difficult to form functional neurosurgery in the body.

Stem cells are into the new environment, differentiate into cells adapting to the signal of new environment for the growth reaction to adjust the new micro-environment. Cultured cells are mixed with co-culture plates, the cells are separated by culture chamber through the upper and lower layers, not in direct contact with the cells. During the culture broth and cytokines pass in and out freely. When the stem cells and somatic cells are co-cultured, morphological and phenotypic changes are often considered to be the merging of two cells to produce new cell type, rather than genuine by the differentiation of stem cells. This co-culture method avoids the problem of cell fusion in differentiation of stem cells. The use of cell-to-cell contact with each other in co-culture induced mechanism creates a micro-environment of the nervous system; cytokines interact interoperably and support each other in the system, which promote bone marrow mesenchymal stem cells to differentiate into nerve cells. Neurotrophic factor secreted from bone marrow mesenchymal stem cells promotes the growth of nerve cells. Bone marrow mesenchymal stem cells placed in a co-culture method mechanism which closer to the micro-environment of the human body and induce differentiation into neural cells, and improved the nutrition and metabolism of the nerve cells, repaired damage of nervous system. The traditional method of isolation and culture of nerve cells has been improved by way of pipetting cell clones gently in our experiments, partially dispersed as much as possible to reduce the mechanical damage, in order to maintain the original link between the cells. Cell death significantly reduced, while clone ball formed faster, closer to the micro-environment of the nervous system in vivo. The final experimental results show that more neural-like protrusions produced and earlier with each other to form a connection in the co-cultured group, and the neuron specific enolase staining positive rate was higher than chemically induced group. Co-cultured bone marrow mesenchymal stem cells were still alive and subculture after two weeks which provides a new method for bone marrow mesenchymal stem cells to differentiate into neural cells. A wealth of experience support was provided for the clinical nervous system disease and injury in cell replacement therapy. In this experiment, the non-induced bone marrow mesenchymal stem cells were also in the neuron specific enolase positive expression and the cells displayed combing with positive cell morphology, prompted the bone marrow mesenchymal stem cells have multilineage differentiation potential, a very small amount of bone marrow mesenchymal stem cells even if not induced, still may be spontaneous differentiation nerve-like cells.

Neuron-specific enolase is an acid soluble protein isolated from brain tissue, one of the enolase isoenzymes, as the specificity recognizing antibody of neurons and neuroendocrine cells, detect the existence of neuronal cells by induction of stem cells, and study the growth of the neuronal development process. Higher positive rate of neuron-specific enolase expression in the ideal induced state, so that the neuron-specific enolase was used as an indicator to detect nerve cells in this experiment. Neuron-specific enolase was used to determine whether the disease involving the nervous system, neuron-specific enolase activity change mainly close to many neurological diseases due to nerve damage^[29], which can be used not only as an indicator to judge the severity of the neuronal damage, but also assess the clinical effect. Neuron-specific enolase positive rate of the nerve cells were observed to determine the effect of differences induced in the experiment.

This experiment compared the effect to induce bone marrow mesenchymal stem cells differentiating into neural cells with two different ways. Experimental results show that genius visible cells shrink smaller into round or triangle in the chemically induced group at day 4, longer dendritic-like cells branch and similar terminal of neurons at the end of the cell were found at 5-6 days, neuron-specific enolase expression was measured after cell morphology was observed, positive rate was not high. Neuron-specific enolase positive expression continued throughout the nerve cell formation stage in the co-culture group. Neuron-specific enolase positive cells could be detected in a higher proportion after a certain period of time by the way of bone marrow mesenchymal stem cells into neural cells, and the positive expression rate is higher than the chemically induced group. Neuron-specific enolase is an acid soluble protein which expresses highly in the nerve cells and neuroendocrine cells as a specific surface marker for mature neurons. The full contact of the two types of cells in the co-culture group, which can maintain contact and signal fully with the proliferation split of the bone marrow mesenchymal stem cells. The characteristics of proliferation and differentiation and survival rate of bone marrow mesenchymal stem cells keep constant after the passage, and maintenance of the nervous cell state better, while promoting the early maturation of neurons. This experiment prompted that the inducing conditions taken improved the proportion of neuron-like cell differentiation to a certain extent with the co-culture way.

Bone marrow mesenchymal stem cells can be differentiated into nerve cells depending on the local environment transplantation in vivo after implantation contact with local cells plays a decisive impact. Sources of human nerve cells are more difficult, so that there are great difficulties in the co-culture method to practical use. Certain joint cytokine combination of this method is better control, more secure, more closely tied to the utility. Co-culture with nerve cells is a breakthrough in the study to find specific cytokine combinations steps. It should also order a thorough understanding of the bone marrow mesenchymal stem cells biological characteristics, transcriptomic, proteomic and molecular level. Now for bone marrow mesenchymal stem cells characteristics are most validated in vitro. it is unclear that what kind of response will produce with the regulation and control of the complex humoral factors in vivo, gene expression changes of induce to differentiation should be transferred to focused on in vivo in the future and the precise mechanism of the signal transduction of exogenous gene to ensure the stability of the recombinant gene in the cell expansion, how to promote the transplanted stem cells to differentiate into specified neurons and glial cells, and to achieve integration with the host brain structure and function, are the most fundamental problems to be solved with the application of stem cell transplantation in the treatment of stroke^[30-36].

诱导剂共培养：谁更适宜骨髓间充质干细胞向神经细胞的分化？

Induction ways of bone marrow mesenchymal stem cells differentiating into nerve cells

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