体外微环境调控诱导人源性骨髓间充质干细胞向胰岛素分泌细胞分化及移植研究
摘要
[研究背景]
糖尿病是严重危害人类健康的常见病和多发病。糖尿病的发病机制至今尚未完全阐明。普遍观点认为糖尿病人群中约5％-10％为1型糖尿病，主要病因是自身免疫紊乱导致的胰岛β细胞选择性破坏，呈胰岛素依赖性；2型糖尿病发病主要与外周胰岛素抵抗有关，晚期存在胰岛素相对不足，其中20％-30％也呈胰岛素依赖性。尽管外源性胰岛素使用控制血糖已经取得很大的进步，但由于其脉冲式的注入不能保持血糖动态平稳，导致大部分糖尿病患者最终会发生各种微血管并发症。功能β细胞移植(全胰移植或胰岛移植)成为治疗胰岛素依赖性糖尿病非常有前景的方法之一。然而，供体的严重缺乏及异体移植导致的免疫排斥反应严重阻碍了其广泛开展，我们希望能找到一种新的方法解决这些问题。
2001年Lumelsky等及Assady分别报道可以将小鼠及人的胚胎干细胞诱导分化为能分泌胰岛素的细胞，由此诱导干细胞分化为新的功能胰岛细胞成为解决胰岛移植供体紧缺新的发展方向之一。但胚胎干细胞存在伦理学限制，同时资源有限，且移植后仍存在异体移植免疫排斥。
成人骨髓间充质干细胞(BM-MSCs)具有多向分化潜能，可向内、中、外三胚层细胞分化，且来源取材方便，资源丰富，并可以实现自体移植，是糖尿病β细胞替代治疗的理想种子细胞。但目前对于BM-MSCs胰岛样分化研究还存在一定缺陷，1)大部分研究采用鼠BM-MSCs，且诱导细胞的胰岛素分泌量少，只有约正常胰岛的1／80，低于胚胎干细胞及神经干细胞诱导后的1／50的胰岛素释放量。2)对于人源性BM-MSCs，其胰岛样分化的研究较少，而且未进行体内的功能鉴定，胰岛素的分泌量及诱导效率均较低。
本课题在上述实验的基础上，利用Percoll密度梯度离心结合贴壁筛选分离并培养扩增12株成人BM-MSCs，并进一步通过建立体外三步法微环境调控诱导BM-MSCs向胰岛样细胞分化。在研究过程中我们发现，1)采用Percoll密度梯度离心结合贴壁筛选法分离得到的细胞在体外可以稳定传代，形态学检测、流式细胞表型及分化能力鉴定均符合国际间充质干细胞特点，且没有明显成瘤性。2)我们意外发现，未诱导的BM-MSCs表达部分胰腺发育相关基因，并在传代后仍能保持特性，这部分研究未见国际报道，提示BM-MSCs存在胰岛前体细胞性质干细胞，具有胰岛样分化潜能。3)我们结合已有研究多次探索，建立了三步法微环境调控诱导方法，成人BM-MSCs在诱导后可以自发形成胰岛样结构，并且多个胰岛相关基因表达激活或增强，DTZ染色及免疫荧光均证实诱导后细胞表达胰岛素及其他内分泌激素，且电镜显示具有胰岛内分泌细胞超微结构。更重要的是，体外高糖刺激试验显示诱导后细胞释放胰岛素，且随着葡萄糖浓度升高而增加，23mM刺激下胰岛素释放量达正常胰岛的1／50，高于已有鼠BM-MSCs诱导后胰岛素释放量60％。而且最重要的是，诱导后细胞移植可以降低糖尿病鼠血糖，改善其糖尿病症状，增加体重，延长生存时间。本研究为人源性BM-MSCs诱导向胰岛素分泌细胞分化替代功能p细胞移植治疗糖尿病，缓解供体紧缺奠定了实验基础。
第一部分：成人BM-MSCs的分离纯化培养及生物学特性鉴定
目的：建立成人BM-MSCs体外分离、培养、扩增的有效方法，并探讨其部分生物学特性、分化潜能及成瘤性。
方法：与血液科骨髓室联系，获取非白血病人骨髓2-3ml，肝素化后利用密度为1．073的Pereoll分离液密度梯度离心，吸取上、中层液体界面处的单个核细胞，结合间充质细胞贴壁生长的特点，通过更换培养液不断纯化分离BM-MSCs并扩增培养。通过倒置显微镜观察细胞的一般形态结构；建立生长曲线了解其生长特性；RT-PCR检测干细胞标志物Oct-4在1-4代成人BM-MSCs的表达；流式细胞学检测CD45，CD34，CD44，CD29，CD14，HLA-DR，CD105等表面分子标志的表达情况；采用含有胰岛素、地塞米松、Ham F12添加剂等的培养基孵育21天诱导成人BM-MSCs向脂肪细胞分化，油红染色鉴定其分化效率；采用PANC-1细胞为阳性对照，软琼脂克隆及裸鼠成瘤实验鉴定BM-MSCs的成瘤性。
结果：原代接种48小时出现贴壁细胞，5-7天后细胞增殖速度明显增快，12-15天原代细胞汇合达80％-90％，原代及传代细胞形态学观察均成典型的成纤维样形态特征，旋涡状生长；传代培养细胞在5-8天时增殖速度最快，8-9天时达到平台期，单代细胞可扩增20-25倍。三个不同供体来源的1-4代BM-MSCs均表达干细胞标志Oct-4，且流式检测显示CD45、CD34、CD14、HLA-DR阴性，CD44、CD29和CD105阳性。经过特定方案诱导后，BM-MSCs细胞变圆，呈现脂肪油滴，油红染色成红色。体外软琼脂克隆BM-MSCs组未见明显克隆细胞团形成，体内裸鼠成瘤实验BM-MSCs也未形成移植瘤。
第二部分：未诱导的成人BM-MSCs胰腺发育相关标记物表达研究
目的：在mRNA及蛋白表达水平检测体外培养的未诱导的成人BM-MSCs胰腺发育相关基因表达情况，探索其胰岛样细胞分化潜能。
方法：采用RT-PCR检测体外培养的未诱导的成人BM-MSCs胰腺发育相关基因ISL-1、Beta2／NeuroD、Nkx6．1、Glut2、PDX-1、Nestin、CK18和CK19在mRNA水平表达情况：Western Blot及免疫组化染色检测体外培养的未诱导的成人BM-MSCs胰腺发育相关基因Glut2、ISL-1、CK18及CK19在蛋白水平及细胞原位表达情况。
结果：RT-PCR显示ISL-1，Beta2／NeuroD，Nkx6．1,Glut2，CK18及CK19均阳性，但PDX-1及Nestin阴性；而Western Blot检测CK18和CK19均阳性，但ISL-1及Glut2阴性；免疫组化显示大部分细胞CK18和CK19阳性，而只有小部分细胞ISL-1及Glut2阳性。
第三部分：体外微环境调控诱导人BM-MSCs分化为胰岛素分泌细胞
目的：建立体外三步法微环境调控诱导成人BM-MSCs向胰岛样细胞分化方案，鉴定分化后细胞胰岛相关基因表达及功能。
方法：采用传代扩增3-5代的成人BM-MSCs，2-3×10~4个细胞／cm~2密度接种，先后利用高糖、碱性成纤维细胞生长因子(bFGF)、尼克酰胺(nicotinamide)、表皮生长因子(EGF)、exendin-4、B27和N2添加剂、肝细胞生长因子(HGF)、activin-A等多种细胞因子及营养因子建立三步法体外微环境调控方案诱导处理。倒置显微镜每日观察诱导后细胞形态的变化；并在每个诱导阶段末，提取诱导及RNA和蛋白检测其胰岛相关基因表达改变，以未诱导细胞细胞为对照；三阶段诱导结束后，双硫腙(Dithizone：DTZ)染色鉴定胰岛素分泌细胞形成情况：免疫荧光检测胰岛素、c肽及胰高血糖素在诱导后细胞中的表达情况；电镜观察细胞超微结构变化；体外高糖刺激实验测定诱导前、诱导中期及诱导末细胞胰岛素分泌能力及其对葡萄糖的应激反应情况。
结果：未诱导细胞传代后仍呈成纤维样，第一阶段结束后细胞呈圆形或不规则形，且易汇聚成团，第二阶段诱导后，细胞逐渐增殖，少量细胞重现梭样形态，汇聚细胞团逐渐增大增多，三阶段诱导结束细胞团体积也较前明显增大，并出现较明显的边界，与胰岛结构相似。DTZ染色显示，诱导形成的细胞团可染成猩红色。RT-PCR及WesternBlot检测显示胰岛相关基因ISL-1、Glut2、PDX-1、Nestin及胰岛素、胰高血糖素在在诱导过程中表达逐渐增强或激活。免疫荧光显示诱导后部分细胞表达胰岛素、c肽及胰高血糖素。电镜检测显示诱导后细胞浆内存在含有圆形或长杆形包装颗粒的空泡状结构，并具有丰富的内质网及线粒体：体外高糖刺激实验显示诱导后的细胞具有胰岛素分泌能力，且分泌量随着葡萄糖浓度升高而增加；23mM高糖刺激下，胰岛素分泌量达到正常胰岛的1／50。
第四部分：成人BM-MSCs诱导分化为胰岛素分泌细胞的体内研究
目的：检测成人BM-MSCs体外诱导分化后的形成的胰岛素分泌细胞移植对纠正1型糖尿病鼠血糖波动的作用，探索其糖尿病治疗效果。
方法：通过腹腔注射大剂量链脲佐菌素(STZ)诱导免疫缺陷裸鼠建立1型糖尿病模型，以制模后一周内两次血糖大于18mmol／l为成模标准。将成模鼠分两组：分别移植诱导后及未诱导成人BM-MSCs于模型鼠肾包膜下，移植细胞量约每只糖尿病鼠0．8-1×10~7细胞。并以未注射STZ诱导糖尿病的正常裸鼠移植PBS为空白对照，连续监测直至移植后30天。每五天通过剪鼠尾取血测空腹血糖，观察统计不同组移植前后血糖、体重变化及存活时间。于移植后2天及30天取移植物HE染色检测不同时间的状态；30天后取移植物胰岛素染色检测。
结果：STZ诱导后裸鼠血糖明显升高，体重减轻，出现典型糖尿病状态，成模率达到80％；细胞移植后可见移植诱导后人BM-MSCs组模型鼠与未诱导人BM-MSCs组相比，血糖明显下降，体重上升，且生存时间明显延长，但未达到正常小鼠水平。HE染色可见移植物在肾包膜下呈小丘样细胞团块；移植后2天移植物HE染色可见细胞边界较模糊，有炎症细胞浸润，没有明显血供形成；移植后30天移植物，有明显血供，且细胞边界清晰。胰岛素染色见有少量细胞呈阳性。
结论：
1，采用Pereoll密度梯度离心结合贴壁筛选法，可以分离纯化人骨髓间充质干细胞，获得的细胞纯度高、扩增能力强，体外扩增12代以上可以维持其幼稚细胞形态特征。
2，培养扩增的成人BM-MSCs干细胞标记物阳性，具有干细胞特性，具有脂肪分化能力，且未见成瘤性。
3，体外培养的未诱导的成人BM-MSCs中部分细胞表达多个胰腺发育相关基因，提示其具有胰岛样细胞分化潜能；且扩增的BM-MSCs存在异质性，可能包含有部分细胞具有胰腺前体细胞的特性；
4，成人BM-MSCs在体外通过三步法微环境调控诱导，可以分化为胰岛样细胞，具有胰岛类似的结构、超微结构，具有受葡萄糖调控的胰岛素分泌功能。
5，诱导后成人BM-MSCs在体内可以有效调控血糖，减轻糖尿病症状，延长生存时间，对糖尿病有明显的治疗作用，提示骨髓间充质干细胞可成为功能B细胞替代细胞来源用于糖尿病治疗。
Background
Diabetes mellitus (DM), which affects millions of people worldwide, is characterized by abnormally high levels of glucose in blood caused by either absolute (type 1 DM) or relative (type 2 DM) insulin deficiency due to the destruction of pancreaticβ-cells by T cells of the immune system or decreased insulin sensitivity respectively. In both types, an inadequate mass of functional islet cells is the major problem for the onset of hyperglycemia and the development of overt diabetes. Islet cell replacement is considered as the optimal treatment recently. However the deficiency of transplantable donor islets has historically hampered its further development.
Lots of researchers have made great effort to expand pancreatic islets by stimulating endogenous regeneration of islets or generating islet-like cells from various sources of stem cells including embryonic stem cells (ESCs), multipotent stem cells within nonendocrine compartments of the pancreas, and various tissue precursor cells, such as hepatic oval cells, splenocytes. However even with these inspiring experimental advances, some obstacles, such as immune rejection, limited sources of stem cells from these organs, still remain.
In the last decades, a new type of multipotent stem cells in adult bone marrow was reported. Bone marrow (BM) is a complex tissue containing two major stem cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Both of them are thought to be multipotent and can be induced to several cell types. Moreover BM-MSCs even can be induced to differentiate into ectodermal neural and epidermal-like cells and endodermal hepatic cells or maybe even other cell lineages from all three germ layers under appropriate experimental conditions. Comparing with other stem cells mentioned above, human BM-MSCs have several obvious advantages: it can be expanded in culture for many doublings, which provides a potentially unlimited source; it has a simple reliable and repeatable procedure to obtain, which makes autogenic transplantation come true and alleviates the major limitations of availability and allogenic rejection simultaneously. Most importantly, there is no need to consider the ethical problems, the major problem of ESCs. In the light of the actual worldwide diabetes epidemic, the generation of new insulin producing cells (IPCs) from human BM-MSCs would offer a particularly useful candidate for deficient donor islets.
In the last 3 years, several studies have discussed the possibility of adult MSCs in BM to differentiate into IPCs with totally different induction protocols. However these articles mostly focus on murine or rat derived BM-MSCs, theβcell differentiation potential and characteristics of human BM-MSCs are poorly documented. Recently there have been three inspiring reports on human BM-MSCs expressing insulin under genetic modulation, and in the present study, we demonstrate that, under defined culture conditions, human BM-MSCs positive for the stem-cell marker Oct-4 express a series of pancreas developing markers including ISL-1, Beta2/NeuroD, Nkx6.1,Glut2, CK18 and CK19 without any induction, and can be induced to form islet-like aggregates morphologically. These aggregates can secrete insulin in a glucose related manner and express upregulated pancreatic developmental genes especially Nestin and PDX-1. Most importantly, after transplanted under the renal capsule of mice, the differentiated cells can decrease the blood glucose and rescue the diabetic mice.
The 1st part: Isolation and cultivation of human BM-MSCs and biological features of the derived cells
Objective: To establish a method for isolation, cultivation and expansion of adult human BM-MSCs, and study their biological features and potential to form carcinoma
Method: 2-3 ml of heparinized adult bone marrow from nonleukemia persons were washed with PBS, and purified by Percoll gradient. Mononuclear cells at the Percoll interface were collected and seeded in expansion medium consisted of DMEM-LG supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The BM-MSCs were purified by their adhesiveness. The changes of cell's morphology were observed by phase contrast microscopy every day, and their expansion fashion at passage 3 was revealed by growth curve. Their expressions of stem cell marker Oct-4 were examined by RT-PCR, and cell surface markers were detected by Flow Cytometry. Their differentiation potential was examined by adipose induction, and their potential to form carcinoma were detected by soft agar colony formation assay and in vivo carcinoma formation assay in athymic nu/nu mice.
Result: Both primary and passage human BM-MSCs showed typically fibroblast- like morphology and had maintined highly proliferative capacity. The cells expressed mRNA of Oct-4, were negative for CD45, CD34, CD14, HLA-DR, and positive for D44, CD29, CD105. The cells showed adipose differentiation after 21 days' induction and no potential to form carcinoma both soft agar colony formation assay and in vivo carcinoma formation assay in athymic nu/nu mice.
The 2nd part: Expression of pancreas developing markers on undifferentiated human BM-MSCs
Objective: To detect the expression of pancreas developing markers on undifferentiated human BM-MSCs in both mRNA and protein level. To reveal their potential to differentiate into insulin producing cells.
Method: The mRNA expressions of pancreas developing markers including ISL-1、Beta2/NeuroD、Nkx6.1、Glut2、PDX-1、Nestin、CK18 and CK19 were detected by RT-PCR. The protein expressions of Glut2、ISL-1、CK18 and CK19 were further examined by Western Blot and immunocytochemistry.
Result: Undifferentiated human BM-MSCs were positive for mRNA of ISL-1, Beta2/NeuroD, Nkx6.1, Glut2, CK18 and CK19. Western Blot showed that derived cells expressed CK18 and CK19, but did not express ISL-1 and Glut2. Immunocytochemistry confirmed most cells were positive for CK18 and CK19, and a small portion of cells were positive for ISL-1 and Glut2.
The 3rd part: Differentiation of human BM-MSCs to insulin producing cells under microenvironmental manipulation in vitro.
Objective: To found a three-stage induction protocol by microenvironmental manipulation to induce human BM-MSCs to differentiate into insulin producing cells in vitro. To reveal the possibility of human BM-MSCs to differentiate into islet-like cells without gene manipulation.
Method: Human BM-MSCs at passage 3-5 were reseeded at a destiny of 2-3×10~4 cells per cm~2, and were treated sequentially by three-stage induction protocol composed of high glucose, bFGF, nicotinamide, EGF, exendin-4, B27, N2, HGF and activin-A et al. The changes of cell's morphology were observed by phase contrast microscopy every day. The mRNA and protein expression profiles of islet related markers were examined by RT-PCR and Western Blot respectively at the end of every induction stage. After three-stage induction, DTZ staining and immunocytochemistry were carried out to examine the insulin expression. Electronic microscopy was used to show the ultrastructure characteristics of differentiated cells and in vitro glucose stimulation was carried out for cells without induction, cells at the end of the second stage and cells after the entire induction.
Results: Induced human BM-MSCs were found to form typical islet-like aggregates and ultrastructure characteristics of matureβcells were proved by electronic microscopy. They were also activated or upregulated to express multiple genes related to pancreaticβcell development and function (PDX-1, Nestin, pax-6, Glut2, ISL-1 and insulin). Insulin c-peptide and glucagon production were identified by immunocytochemistry. Insulin expression was further confirmed by DTZ staining and in vitro glucose stimulation which certified as a glucose related manner.
The 4th part: Transplantation of insulin producing cells differentiated from human BM-MSCs can control diabetes of STZ induced diabetic nude mice.
Objective: To investigate the in vivo effect of insulin producing cells differentiated from human BM-MSCs by microenvironmental manipulation on diabetes controls in nude mice
Method: The diabetic nude mice were achieved by STZ (220 mg/kg) injection as blood glucose level was higher than 18mmol/l. Treated and nontreated human BM-MSCs were transplanted under the renal capsule of diabetic nude mice respectively. Blood glucose levels, weight and survival time were monitored for 30 days after transplantation. The graft status was examined by HE staining 2 days after transplantation and at the end of the observation period. Immunocytochemistry was carried out to detect the insulin expression.
Result: Xenotransplantation of insulin producing cells from human BM-MSCs can decrease the hyperglycemia and prolong the survival time of STZ induced diabetic nude mice.
Conclusion:
1. Human BM-MSCs can be isolated and purified by Percoll gradient combined with adhesiveness selection. Derived cells can be cultured stably for 12 passages and expanded quickly.
2. Expanded human BM-MSCs have the stem cell characteristics. They are positive for stem cell marker- Oct-4, and have the potential to differentiate to adipose cells.
3. Expanded human BM-MSCs expresse several pancreas developing markers, indicating that they have the potential to differentiate into insulin producing cells.
4. Human BM-MSCs can be induced into insulin producing cells in vitro by microenvironmental manipulation with a three-stage protocol. Differentiated cells have islet-like morphology and function.
5. Differentiated cells transplantation can control diabetes of STZ induce nude mice, indicating human BM-MSCs can be another source of functionalβcells for diabetes treatment.
糖尿病是严重危害人类健康的常见病和多发病。糖尿病的发病机制至今尚未完全阐明。普遍观点认为糖尿病人群中约5％-10％为1型糖尿病，主要病因是自身免疫紊乱导致的胰岛β细胞选择性破坏，呈胰岛素依赖性；2型糖尿病发病主要与外周胰岛素抵抗有关，晚期存在胰岛素相对不足，其中20％-30％也呈胰岛素依赖性。尽管外源性胰岛素使用控制血糖已经取得很大的进步，但由于其脉冲式的注入不能保持血糖动态平稳，导致大部分糖尿病患者最终会发生各种微血管并发症。功能β细胞移植(全胰移植或胰岛移植)成为治疗胰岛素依赖性糖尿病非常有前景的方法之一。然而，供体的严重缺乏及异体移植导致的免疫排斥反应严重阻碍了其广泛开展，我们希望能找到一种新的方法解决这些问题。
2001年Lumelsky等及Assady分别报道可以将小鼠及人的胚胎干细胞诱导分化为能分泌胰岛素的细胞，由此诱导干细胞分化为新的功能胰岛细胞成为解决胰岛移植供体紧缺新的发展方向之一。但胚胎干细胞存在伦理学限制，同时资源有限，且移植后仍存在异体移植免疫排斥。
成人骨髓间充质干细胞(BM-MSCs)具有多向分化潜能，可向内、中、外三胚层细胞分化，且来源取材方便，资源丰富，并可以实现自体移植，是糖尿病β细胞替代治疗的理想种子细胞。但目前对于BM-MSCs胰岛样分化研究还存在一定缺陷，1)大部分研究采用鼠BM-MSCs，且诱导细胞的胰岛素分泌量少，只有约正常胰岛的1／80，低于胚胎干细胞及神经干细胞诱导后的1／50的胰岛素释放量。2)对于人源性BM-MSCs，其胰岛样分化的研究较少，而且未进行体内的功能鉴定，胰岛素的分泌量及诱导效率均较低。
本课题在上述实验的基础上，利用Percoll密度梯度离心结合贴壁筛选分离并培养扩增12株成人BM-MSCs，并进一步通过建立体外三步法微环境调控诱导BM-MSCs向胰岛样细胞分化。在研究过程中我们发现，1)采用Percoll密度梯度离心结合贴壁筛选法分离得到的细胞在体外可以稳定传代，形态学检测、流式细胞表型及分化能力鉴定均符合国际间充质干细胞特点，且没有明显成瘤性。2)我们意外发现，未诱导的BM-MSCs表达部分胰腺发育相关基因，并在传代后仍能保持特性，这部分研究未见国际报道，提示BM-MSCs存在胰岛前体细胞性质干细胞，具有胰岛样分化潜能。3)我们结合已有研究多次探索，建立了三步法微环境调控诱导方法，成人BM-MSCs在诱导后可以自发形成胰岛样结构，并且多个胰岛相关基因表达激活或增强，DTZ染色及免疫荧光均证实诱导后细胞表达胰岛素及其他内分泌激素，且电镜显示具有胰岛内分泌细胞超微结构。更重要的是，体外高糖刺激试验显示诱导后细胞释放胰岛素，且随着葡萄糖浓度升高而增加，23mM刺激下胰岛素释放量达正常胰岛的1／50，高于已有鼠BM-MSCs诱导后胰岛素释放量60％。而且最重要的是，诱导后细胞移植可以降低糖尿病鼠血糖，改善其糖尿病症状，增加体重，延长生存时间。本研究为人源性BM-MSCs诱导向胰岛素分泌细胞分化替代功能p细胞移植治疗糖尿病，缓解供体紧缺奠定了实验基础。
第一部分：成人BM-MSCs的分离纯化培养及生物学特性鉴定
目的：建立成人BM-MSCs体外分离、培养、扩增的有效方法，并探讨其部分生物学特性、分化潜能及成瘤性。
方法：与血液科骨髓室联系，获取非白血病人骨髓2-3ml，肝素化后利用密度为1．073的Pereoll分离液密度梯度离心，吸取上、中层液体界面处的单个核细胞，结合间充质细胞贴壁生长的特点，通过更换培养液不断纯化分离BM-MSCs并扩增培养。通过倒置显微镜观察细胞的一般形态结构；建立生长曲线了解其生长特性；RT-PCR检测干细胞标志物Oct-4在1-4代成人BM-MSCs的表达；流式细胞学检测CD45，CD34，CD44，CD29，CD14，HLA-DR，CD105等表面分子标志的表达情况；采用含有胰岛素、地塞米松、Ham F12添加剂等的培养基孵育21天诱导成人BM-MSCs向脂肪细胞分化，油红染色鉴定其分化效率；采用PANC-1细胞为阳性对照，软琼脂克隆及裸鼠成瘤实验鉴定BM-MSCs的成瘤性。
结果：原代接种48小时出现贴壁细胞，5-7天后细胞增殖速度明显增快，12-15天原代细胞汇合达80％-90％，原代及传代细胞形态学观察均成典型的成纤维样形态特征，旋涡状生长；传代培养细胞在5-8天时增殖速度最快，8-9天时达到平台期，单代细胞可扩增20-25倍。三个不同供体来源的1-4代BM-MSCs均表达干细胞标志Oct-4，且流式检测显示CD45、CD34、CD14、HLA-DR阴性，CD44、CD29和CD105阳性。经过特定方案诱导后，BM-MSCs细胞变圆，呈现脂肪油滴，油红染色成红色。体外软琼脂克隆BM-MSCs组未见明显克隆细胞团形成，体内裸鼠成瘤实验BM-MSCs也未形成移植瘤。
第二部分：未诱导的成人BM-MSCs胰腺发育相关标记物表达研究
目的：在mRNA及蛋白表达水平检测体外培养的未诱导的成人BM-MSCs胰腺发育相关基因表达情况，探索其胰岛样细胞分化潜能。
方法：采用RT-PCR检测体外培养的未诱导的成人BM-MSCs胰腺发育相关基因ISL-1、Beta2／NeuroD、Nkx6．1、Glut2、PDX-1、Nestin、CK18和CK19在mRNA水平表达情况：Western Blot及免疫组化染色检测体外培养的未诱导的成人BM-MSCs胰腺发育相关基因Glut2、ISL-1、CK18及CK19在蛋白水平及细胞原位表达情况。
结果：RT-PCR显示ISL-1，Beta2／NeuroD，Nkx6．1,Glut2，CK18及CK19均阳性，但PDX-1及Nestin阴性；而Western Blot检测CK18和CK19均阳性，但ISL-1及Glut2阴性；免疫组化显示大部分细胞CK18和CK19阳性，而只有小部分细胞ISL-1及Glut2阳性。
第三部分：体外微环境调控诱导人BM-MSCs分化为胰岛素分泌细胞
目的：建立体外三步法微环境调控诱导成人BM-MSCs向胰岛样细胞分化方案，鉴定分化后细胞胰岛相关基因表达及功能。
方法：采用传代扩增3-5代的成人BM-MSCs，2-3×10~4个细胞／cm~2密度接种，先后利用高糖、碱性成纤维细胞生长因子(bFGF)、尼克酰胺(nicotinamide)、表皮生长因子(EGF)、exendin-4、B27和N2添加剂、肝细胞生长因子(HGF)、activin-A等多种细胞因子及营养因子建立三步法体外微环境调控方案诱导处理。倒置显微镜每日观察诱导后细胞形态的变化；并在每个诱导阶段末，提取诱导及RNA和蛋白检测其胰岛相关基因表达改变，以未诱导细胞细胞为对照；三阶段诱导结束后，双硫腙(Dithizone：DTZ)染色鉴定胰岛素分泌细胞形成情况：免疫荧光检测胰岛素、c肽及胰高血糖素在诱导后细胞中的表达情况；电镜观察细胞超微结构变化；体外高糖刺激实验测定诱导前、诱导中期及诱导末细胞胰岛素分泌能力及其对葡萄糖的应激反应情况。
结果：未诱导细胞传代后仍呈成纤维样，第一阶段结束后细胞呈圆形或不规则形，且易汇聚成团，第二阶段诱导后，细胞逐渐增殖，少量细胞重现梭样形态，汇聚细胞团逐渐增大增多，三阶段诱导结束细胞团体积也较前明显增大，并出现较明显的边界，与胰岛结构相似。DTZ染色显示，诱导形成的细胞团可染成猩红色。RT-PCR及WesternBlot检测显示胰岛相关基因ISL-1、Glut2、PDX-1、Nestin及胰岛素、胰高血糖素在在诱导过程中表达逐渐增强或激活。免疫荧光显示诱导后部分细胞表达胰岛素、c肽及胰高血糖素。电镜检测显示诱导后细胞浆内存在含有圆形或长杆形包装颗粒的空泡状结构，并具有丰富的内质网及线粒体：体外高糖刺激实验显示诱导后的细胞具有胰岛素分泌能力，且分泌量随着葡萄糖浓度升高而增加；23mM高糖刺激下，胰岛素分泌量达到正常胰岛的1／50。
第四部分：成人BM-MSCs诱导分化为胰岛素分泌细胞的体内研究
目的：检测成人BM-MSCs体外诱导分化后的形成的胰岛素分泌细胞移植对纠正1型糖尿病鼠血糖波动的作用，探索其糖尿病治疗效果。
方法：通过腹腔注射大剂量链脲佐菌素(STZ)诱导免疫缺陷裸鼠建立1型糖尿病模型，以制模后一周内两次血糖大于18mmol／l为成模标准。将成模鼠分两组：分别移植诱导后及未诱导成人BM-MSCs于模型鼠肾包膜下，移植细胞量约每只糖尿病鼠0．8-1×10~7细胞。并以未注射STZ诱导糖尿病的正常裸鼠移植PBS为空白对照，连续监测直至移植后30天。每五天通过剪鼠尾取血测空腹血糖，观察统计不同组移植前后血糖、体重变化及存活时间。于移植后2天及30天取移植物HE染色检测不同时间的状态；30天后取移植物胰岛素染色检测。
结果：STZ诱导后裸鼠血糖明显升高，体重减轻，出现典型糖尿病状态，成模率达到80％；细胞移植后可见移植诱导后人BM-MSCs组模型鼠与未诱导人BM-MSCs组相比，血糖明显下降，体重上升，且生存时间明显延长，但未达到正常小鼠水平。HE染色可见移植物在肾包膜下呈小丘样细胞团块；移植后2天移植物HE染色可见细胞边界较模糊，有炎症细胞浸润，没有明显血供形成；移植后30天移植物，有明显血供，且细胞边界清晰。胰岛素染色见有少量细胞呈阳性。
结论：
1，采用Pereoll密度梯度离心结合贴壁筛选法，可以分离纯化人骨髓间充质干细胞，获得的细胞纯度高、扩增能力强，体外扩增12代以上可以维持其幼稚细胞形态特征。
2，培养扩增的成人BM-MSCs干细胞标记物阳性，具有干细胞特性，具有脂肪分化能力，且未见成瘤性。
3，体外培养的未诱导的成人BM-MSCs中部分细胞表达多个胰腺发育相关基因，提示其具有胰岛样细胞分化潜能；且扩增的BM-MSCs存在异质性，可能包含有部分细胞具有胰腺前体细胞的特性；
4，成人BM-MSCs在体外通过三步法微环境调控诱导，可以分化为胰岛样细胞，具有胰岛类似的结构、超微结构，具有受葡萄糖调控的胰岛素分泌功能。
5，诱导后成人BM-MSCs在体内可以有效调控血糖，减轻糖尿病症状，延长生存时间，对糖尿病有明显的治疗作用，提示骨髓间充质干细胞可成为功能B细胞替代细胞来源用于糖尿病治疗。
Background
Diabetes mellitus (DM), which affects millions of people worldwide, is characterized by abnormally high levels of glucose in blood caused by either absolute (type 1 DM) or relative (type 2 DM) insulin deficiency due to the destruction of pancreaticβ-cells by T cells of the immune system or decreased insulin sensitivity respectively. In both types, an inadequate mass of functional islet cells is the major problem for the onset of hyperglycemia and the development of overt diabetes. Islet cell replacement is considered as the optimal treatment recently. However the deficiency of transplantable donor islets has historically hampered its further development.
Lots of researchers have made great effort to expand pancreatic islets by stimulating endogenous regeneration of islets or generating islet-like cells from various sources of stem cells including embryonic stem cells (ESCs), multipotent stem cells within nonendocrine compartments of the pancreas, and various tissue precursor cells, such as hepatic oval cells, splenocytes. However even with these inspiring experimental advances, some obstacles, such as immune rejection, limited sources of stem cells from these organs, still remain.
In the last decades, a new type of multipotent stem cells in adult bone marrow was reported. Bone marrow (BM) is a complex tissue containing two major stem cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Both of them are thought to be multipotent and can be induced to several cell types. Moreover BM-MSCs even can be induced to differentiate into ectodermal neural and epidermal-like cells and endodermal hepatic cells or maybe even other cell lineages from all three germ layers under appropriate experimental conditions. Comparing with other stem cells mentioned above, human BM-MSCs have several obvious advantages: it can be expanded in culture for many doublings, which provides a potentially unlimited source; it has a simple reliable and repeatable procedure to obtain, which makes autogenic transplantation come true and alleviates the major limitations of availability and allogenic rejection simultaneously. Most importantly, there is no need to consider the ethical problems, the major problem of ESCs. In the light of the actual worldwide diabetes epidemic, the generation of new insulin producing cells (IPCs) from human BM-MSCs would offer a particularly useful candidate for deficient donor islets.
In the last 3 years, several studies have discussed the possibility of adult MSCs in BM to differentiate into IPCs with totally different induction protocols. However these articles mostly focus on murine or rat derived BM-MSCs, theβcell differentiation potential and characteristics of human BM-MSCs are poorly documented. Recently there have been three inspiring reports on human BM-MSCs expressing insulin under genetic modulation, and in the present study, we demonstrate that, under defined culture conditions, human BM-MSCs positive for the stem-cell marker Oct-4 express a series of pancreas developing markers including ISL-1, Beta2/NeuroD, Nkx6.1,Glut2, CK18 and CK19 without any induction, and can be induced to form islet-like aggregates morphologically. These aggregates can secrete insulin in a glucose related manner and express upregulated pancreatic developmental genes especially Nestin and PDX-1. Most importantly, after transplanted under the renal capsule of mice, the differentiated cells can decrease the blood glucose and rescue the diabetic mice.
The 1st part: Isolation and cultivation of human BM-MSCs and biological features of the derived cells
Objective: To establish a method for isolation, cultivation and expansion of adult human BM-MSCs, and study their biological features and potential to form carcinoma
Method: 2-3 ml of heparinized adult bone marrow from nonleukemia persons were washed with PBS, and purified by Percoll gradient. Mononuclear cells at the Percoll interface were collected and seeded in expansion medium consisted of DMEM-LG supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The BM-MSCs were purified by their adhesiveness. The changes of cell's morphology were observed by phase contrast microscopy every day, and their expansion fashion at passage 3 was revealed by growth curve. Their expressions of stem cell marker Oct-4 were examined by RT-PCR, and cell surface markers were detected by Flow Cytometry. Their differentiation potential was examined by adipose induction, and their potential to form carcinoma were detected by soft agar colony formation assay and in vivo carcinoma formation assay in athymic nu/nu mice.
Result: Both primary and passage human BM-MSCs showed typically fibroblast- like morphology and had maintined highly proliferative capacity. The cells expressed mRNA of Oct-4, were negative for CD45, CD34, CD14, HLA-DR, and positive for D44, CD29, CD105. The cells showed adipose differentiation after 21 days' induction and no potential to form carcinoma both soft agar colony formation assay and in vivo carcinoma formation assay in athymic nu/nu mice.
The 2nd part: Expression of pancreas developing markers on undifferentiated human BM-MSCs
Objective: To detect the expression of pancreas developing markers on undifferentiated human BM-MSCs in both mRNA and protein level. To reveal their potential to differentiate into insulin producing cells.
Method: The mRNA expressions of pancreas developing markers including ISL-1、Beta2/NeuroD、Nkx6.1、Glut2、PDX-1、Nestin、CK18 and CK19 were detected by RT-PCR. The protein expressions of Glut2、ISL-1、CK18 and CK19 were further examined by Western Blot and immunocytochemistry.
Result: Undifferentiated human BM-MSCs were positive for mRNA of ISL-1, Beta2/NeuroD, Nkx6.1, Glut2, CK18 and CK19. Western Blot showed that derived cells expressed CK18 and CK19, but did not express ISL-1 and Glut2. Immunocytochemistry confirmed most cells were positive for CK18 and CK19, and a small portion of cells were positive for ISL-1 and Glut2.
The 3rd part: Differentiation of human BM-MSCs to insulin producing cells under microenvironmental manipulation in vitro.
Objective: To found a three-stage induction protocol by microenvironmental manipulation to induce human BM-MSCs to differentiate into insulin producing cells in vitro. To reveal the possibility of human BM-MSCs to differentiate into islet-like cells without gene manipulation.
Method: Human BM-MSCs at passage 3-5 were reseeded at a destiny of 2-3×10~4 cells per cm~2, and were treated sequentially by three-stage induction protocol composed of high glucose, bFGF, nicotinamide, EGF, exendin-4, B27, N2, HGF and activin-A et al. The changes of cell's morphology were observed by phase contrast microscopy every day. The mRNA and protein expression profiles of islet related markers were examined by RT-PCR and Western Blot respectively at the end of every induction stage. After three-stage induction, DTZ staining and immunocytochemistry were carried out to examine the insulin expression. Electronic microscopy was used to show the ultrastructure characteristics of differentiated cells and in vitro glucose stimulation was carried out for cells without induction, cells at the end of the second stage and cells after the entire induction.
Results: Induced human BM-MSCs were found to form typical islet-like aggregates and ultrastructure characteristics of matureβcells were proved by electronic microscopy. They were also activated or upregulated to express multiple genes related to pancreaticβcell development and function (PDX-1, Nestin, pax-6, Glut2, ISL-1 and insulin). Insulin c-peptide and glucagon production were identified by immunocytochemistry. Insulin expression was further confirmed by DTZ staining and in vitro glucose stimulation which certified as a glucose related manner.
The 4th part: Transplantation of insulin producing cells differentiated from human BM-MSCs can control diabetes of STZ induced diabetic nude mice.
Objective: To investigate the in vivo effect of insulin producing cells differentiated from human BM-MSCs by microenvironmental manipulation on diabetes controls in nude mice
Method: The diabetic nude mice were achieved by STZ (220 mg/kg) injection as blood glucose level was higher than 18mmol/l. Treated and nontreated human BM-MSCs were transplanted under the renal capsule of diabetic nude mice respectively. Blood glucose levels, weight and survival time were monitored for 30 days after transplantation. The graft status was examined by HE staining 2 days after transplantation and at the end of the observation period. Immunocytochemistry was carried out to detect the insulin expression.
Result: Xenotransplantation of insulin producing cells from human BM-MSCs can decrease the hyperglycemia and prolong the survival time of STZ induced diabetic nude mice.
Conclusion:
1. Human BM-MSCs can be isolated and purified by Percoll gradient combined with adhesiveness selection. Derived cells can be cultured stably for 12 passages and expanded quickly.
2. Expanded human BM-MSCs have the stem cell characteristics. They are positive for stem cell marker- Oct-4, and have the potential to differentiate to adipose cells.
3. Expanded human BM-MSCs expresse several pancreas developing markers, indicating that they have the potential to differentiate into insulin producing cells.
4. Human BM-MSCs can be induced into insulin producing cells in vitro by microenvironmental manipulation with a three-stage protocol. Differentiated cells have islet-like morphology and function.
5. Differentiated cells transplantation can control diabetes of STZ induce nude mice, indicating human BM-MSCs can be another source of functionalβcells for diabetes treatment.
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