背景及意义

在人类颅面综合征疾病谱中，Treacher Collins综合征(Treacher Collins syndrome,TCS)的发生与第一、二鳃弓密切相关。该综合征出现小耳畸形症状的几率约为60%。本课题组曾对国人TCS患者及家属进行临床资料分析及全外显子测序研究，探讨了国人TCS的临床表现及致病基因突变特点。既往研究中，我们发现在征得患者及家属知情同意后单次抽血所获取的适量血液样本难以满足多次、不同种类遗传学检测，而重复获取同一患者血液样本不仅难以实现而且会给患者带来额外损伤。耳郭软骨组织工程对治疗小耳畸形所带来的耳软骨缺损意义重大。作为最常用的种子细胞，患者自体残耳软骨样本量少，易老化，目前需要开辟种子细胞获取的新途径。在针对TCS发病机理的遗传学领域，现有TCS疾病模型难以动态反映人类TCS的发病特征。诱导性多能干细胞（Induced pluripotent stem cells，iPSCs）作为胚胎干细胞（Embryonic stem cells，ESCs）的替代产物，避免了运用ESCs产生的争议，可高度模拟人类发育起点。本课题为既往课题组对TCS研究的延续性课题，针对TCS中小耳畸形这一典型症状，以iPSCs为源头构建可动态观察的TCS软骨体外发育模型具有深刻的意义：① 通过对本实验制备的TCS患者iPSCs细胞系PSHi002-A进行染色体核型以及一代测序检测，观察制备出的iPSCs是否存在染色体畸变以及是否携带与患者自身细胞相同的基因突变信息，明确是否可解决单次血液样本难以满足多次、不同种类的遗传学检测问题；② iPSCs具有全能特性，可长期、稳定、大量增殖。针对患者存在的基因突变，在遵守法规的前提下，未来运用基因治疗技术对实验制备的细胞进行“修正”，获得健康的目的细胞，可作为组织工程种子细胞的新来源，用于相应患者自身组织（软骨、骨等）缺损的修复，避免出现种子细胞不足、移植物排斥反应的发生；③ 利用iPSCs 分化为研究所需的目的细胞，未来可用于药物研发早期阶段的毒性检测试验，极大节约花费在动物及人体层面试验所需要的时间及经费成本。

研究目的

1.制备TCOF1基因突变TCS患者iPSC细胞系PSHi002-A，获得状态稳定、近似胚胎发育早期、包含患者基因突变信息的PSHi002-A，一方面可为疾病遗传学检测获得稳定、足量的细胞样本来源，另一方面为该TCS患者体外软骨发育模型提供“起点”，对后续细胞层面的药物筛选、基因治疗提供支持。

2.利用PSHi002-A诱导分化出神经嵴样细胞（Neural crest-like cells，NCLCs）。模拟胚胎发育早期对颅面部发育至关重要的神经嵴细胞（Neural crest cells，NCCs），为探索NCCs在TCS患者颅面结构形成异常中更深层次的作用提供细胞来源支持。

3.利用制备的患者NCLCs分化出神经嵴来源间充质干细胞（Neural crest-derived mesenchymal stem cells，NCMCs）向软骨方向诱导分化，模拟TCS患者胚胎发育早期由NCCs经历上皮-间充质转化后形成颅面部软骨的过程。

研究方法

1.经中国医学科学院整形外科医院伦理委员会批准，在患者及家属充分知情的前提下，征得该TCS患者及家属同意，签署知情同意书，获取适量患者血液样本，分离培养单核细胞。通过非整合重编程方法制备成iPSCs，在专用培养体系中培养，出现ESCs样克隆后，通过镜下观察细胞形态，碱性磷酸酶检测，流式细胞术鉴定、病原体检测、支原体检测、细胞免疫荧光染色、实时定量反转录聚合酶链式反应（Real-time quantitative reverse transcription polymerase chain reaction，RT-qPCR）检测多能性基因表达、染色体核型测试、畸胎瘤实验鉴定iPSCs是否制备成功，一代测序检测患者iPSCs是否携带患者TCOF1基因突变信息。

2.利用EDTA消化法对患者iPSC细胞系进行传代，体外培养数代，待制备出的患者来源iPSC细胞系状态稳定后，向NCCs方向诱导，获得NCLCs。细胞免疫荧光染色，RT-qPCR鉴定NCLCs相应NCCs标志物的表达情况。

3.将制备出的NCLCs于体外向间充质干细胞（Mesenchymal stem cells，MSCs）方向诱导分化，获得NCMCs。对制备出的NCMCs运用流式细胞术检测MSC表面标志物CD73，CD105，CD45，CD90的表达情况，并对NCMCs进行成脂，成骨，成软骨诱导分化，检测其三系分化能力。

4.制备海藻酸钠-甲基丙烯酸酐化明胶（Gelatin methacryloyl，GelMA）水凝胶，将患者来源NCMCs接种于水凝胶，以3D悬浮方式向软骨方向分化培养，分别对体外培养7天，21天以及56天的细胞水凝胶复合体进行活/死细胞特异染色。取体外培养56天的细胞水凝胶复合体进行阿利新蓝，番红O，甲苯胺蓝特异性染色，观察NCMCs在3D水凝胶培养状态下的成软骨状态。

研究结果

1.镜下PSHi002-A呈克隆状生长，克隆形态包括集落状、表面隆起的圆形或者椭圆形，边界清晰，核质比大，细胞团内部细胞排列紧凑，有层峦叠嶂之感，细胞与细胞形态界限不易区分。iPSCs碱性磷酸酶呈阳性。流式细胞术、细胞免疫荧光染色结果显示iPSCs多能蛋白SSEA4，TRA-1-81，OCT4，TRA-1-60阳性表达。RT-qPCR显示iPSCs多能基因POU5F1，NANOG阳性表达。染色体核型分析未见异常。一代测序结果显示制备的该TCS患者iPSCs与既往课题组对患者所行高通量测序结果所示的突变位点一致。畸胎瘤实验显示制备的iPSCs具有向外胚层、中胚层以及内胚层分化的能力。

2.镜下观察NCLCs形成过程，细胞由排列紧凑呈集落状生长的密集形态逐步分化为较为松散的多角形或星形细胞，核质比减少。细胞免疫荧光以及RT-qPCR结果表明NCLCs表达包括颅NCCs标志物TWIST1基因在内的NCCs标志物，同时表达胚胎颅面早期发育特异基因OTX2，表明高度模拟颅NCCs的NCLCs制备成功。

3.镜下可见由NCLCs分化而成的NCMCs呈纺锤样或长梭形贴壁生长。流式细胞术检测结果显示制备出的NCMCs阳性表达CD73，CD105，CD90 MSC阳性标志物，阴性表达CD45 MSC阴性标志物。成脂、成软骨以及成骨实验显示NCMCs具备三系分化能力。

4.对体外3D培养的NCMCs水凝胶复合体在向软骨诱导分化的过程中进行活/死细胞染色以及软骨特异性染色。结果显示细胞水凝胶复合体中，培养7天，21天时细胞活力较高，细胞团形成增多且体积增大。体外培养56天时，水凝胶中细胞总数目降低。阿利新蓝，番红O，甲苯胺蓝染色表明有软骨样细胞及基质形成。

研究结论

1.PSHi002-A可由患者少量外周血单核细胞成功制备，经相应检测，符合制备标准。PSHi002-A继承了该患者基因突变特点，可为疾病遗传学检测获得稳定、“年轻”的细胞样本来源。PSHi002-A具备成为该TCS患者体外软骨发育模型“起点”的资质。

2.PSHi002-A可分化为表达包括颅NCCs标志物TWIST1基因在内的NCCs标志物的NCLCs。对制备出的NCLCs进行筛选培养，可获得较高阳性表达NCCs标志物的NCLCs。

3.NCLCs可继续分化为具备成脂、成软骨以及成骨能力的NCMCs。此外，该患者来源NCMCs与水凝胶结合，具备在体外3D环境下向软骨分化的能力。

Background

In the human craniofacial syndrome disease spectrum, the occurrence of Treacher Collins syndrome (TCS) is closely related to the first and second branchial arch. The probability of microtia in this syndrome is about 60%. Our group analyzed the clinical data and whole-exome sequencing of Chinese patients with TCS and their families, and discussed the clinical manifestations and pathogenic gene mutation characteristics of TCS in Chinese. In previous study, we found that the appropriate amount of blood samples obtained from a single blood sample after obtaining the informed consent of patients and their families was difficult to meet multiple and different kinds of genetic tests. Repeated acquisition of blood samples from the same patient is not only difficult to achieve, but also brings additional damage to patients. Auricular cartilage tissue engineering is of great significance in the treatment of ear cartilage defects caused by microtia. As the most commonly used seed cells, the sample size of autologous residual ear cartilage is small and easy to aging, so it is necessary to open up a new way to obtain seed cells. In the field of genetics aiming at the pathogenesis of TCS, the existing TCS disease models are difficult to dynamically reflect the characteristics of human TCS. As a substitute of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) avoid the controversy caused by the use of ESCs and can highly simulate the starting point of human development. This subject is a continuous subject of the previous research group on TCS. Aiming at the typical symptom of TCS microtia, it is of profound significance to construct a dynamically observable TCS cartilage development model in vitro with iPSCs as the source: ① Karyotype detection and first-generation sequencing were used to observe whether the prepared iPSCs has chromosome aberration and whether it carries the same gene mutation information as the patient's own cells, and to determine whether it can solve the problem that a single blood sample is difficult to meet multiple and different kinds of genetic testing. ② iPSCs is totipotent, which can be long-term, stable and proliferated in large quantities. In view of the gene mutation in patients, on the premise of abiding by the laws and regulations, gene therapy technology will be used to "correct" the cells prepared in the experiment in the future to obtain healthy target cells, which can be used as a new source of seed cells for tissue engineering. It can be used to repair the defects of the corresponding patients' own tissues (cartilage, bone, etc.) to avoid the occurrence of seed cell deficiency and graft rejection. ③ The use of iPSCs to differentiate into target cells for research can be used in the toxicity test in the early stage of drug screening in the future, which can greatly save the time and cost spent on animal and human level experiments.

Objective

1. The iPSC cell line PSHi002-An of TCS patients with TCOF1 gene mutation was prepared, and the PSHi002-A with stable state, similar to early embryonic development and containing the information of gene mutation was obtained. On the one hand, it can obtain a stable and sufficient source of cell samples for disease genetic detection, on the other hand, it can provide a "starting point" for the in vitro cartilage development model of this TCS patient, and provide support for future drug screening and gene therapy at the cellular level.

2. Neural crest-like cells (NCLCs) were induced by PSHi002-A. To simulate the neural crest cells (NCCs) which are very important for craniofacial development in the early stage of embryonic development, and to provide cell source support for exploring the deeper role of NCCs in craniofacial structural abnormalities in patients with TCS.

3. The prepared NCLCs was used to differentiate neural crest-derived mesenchymal stem cells (NCMCs) into cartilage to simulate the process of craniofacial cartilage formation after epithelial-mesenchymal transformation from NCCs in the early stage of embryonic development in patients with TCS.

Methods

1. With the approval of the ethics committee of Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, patient and family members with congenital microtia combined with TCS were recruited with the consent of fully informed patients and family members, signed the informed consent form, obtained a suitable number of patient blood samples, and isolated and cultured monocytes. Prepared as iPSCs by a nonintegrating reprogramming method, cultured in a specialized culture system, after the emergence of ESCs like clones, the cells were observed microscopically for morphology, alkaline phosphatase assay, flow cytometry, pathogen detection, mycoplasma assay, immunofluorescence staining of cells,pluripotency gene expression was assessed by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), karyotype testing, and teratoma experiments to determine whether iPSCs were prepared successfully. The first-generation sequencing technique was used to detect whether patients' iPSCs carries TCOF1 gene mutation information.

2. To obtain NCLCs by passaging patient iPSC line using EDTA digestion and culturing them in vitro for several passages, after the status of the patient derived iPSC line to be prepared has stabilized. Cell immunofluorescence staining, RT-qPCR were performed to identify the expression of corresponding markers in NCLCs.

3. The prepared NCLCs was induced to differentiate into MSCs in vitro and NCMCs was obtained. Flow cytometry was used to detect the expression of surface marker CD73,CD105,CD45,CD90 in the prepared NCMCs. The differentiation of NCMCs was induced by adipogenesis, osteogenesis and chondrogenesis, and its three-line differentiation ability was tested.

4. Sodium alginate- gelatin methacryloyl (GelMA) hydrogel was prepared. Patient-derived NCMCs was inoculated into the hydrogel and cultured in 3D suspension to differentiate into cartilage. The cell hydrogel complexes cultured in vitro for 7 days, 21 days and 56 days were stained for living / dead cells. The cellular hydrogel complex cultured in vitro for 56 days was stained with alcian blue, fuchsin O and toluidine blue to observe the chondrogenic state of NCMCs in 3D hydrogel culture.

Results

1. Microscopically, the PSHi002-A showed clonal growth, and the clonal morphology included colony like, round or oval shapes with raised surface, clear boundary, large nucleo cytoplasmic ratio, compact arrangement of cells inside the cell mass, feeling of laminated peaks, and indistinguishable boundary between cell and cell morphology. iPSCs were positive for alkaline phosphatase. The results of flow cytometry and cellular immunofluorescence staining showed that iPSCs pluripotent protein SSEA4, TRA-1-81, OCT4,TRA-1-60 were positive. RT-qPCR showed positive expression of iPSCs multipotent gene POU5F1, NANOG. No abnormality was found in chromosome karyotype analysis. The results of first-generation sequencing showed that the prepared iPSCs of the TCS patients was consistent with the mutation sites shown by the previous high-throughput sequencing results of the patients.Teratoma experiments revealed that the prepared iPSCs had the ability to differentiate into ectoderm, mesoderm as well as endoderm.

2. Microscopically, the formation process of NCLCs was observed, and the cells were gradually differentiated into looser polygonal or astrocytic cells by a dense morphology arranged in compact and colony like growth, and the nuclear cytoplasmic ratio was reduced. The results of cellular immunofluorescence and RT-qPCR showed that NCLCs expressed NCCs markers including TWIST1, a marker of cranial NCCs, and OTX2, a gene specific for early craniofacial development, indicating that the NCLCs that highly mimics craniofacial NCCs was successfully prepared.

3. Microscopically, NCMCs differentiated from NCLCs showed spindle like or long spindle shaped adherent growth. Flow cytometry results showed that the prepared NCMCs expressed positive CD73, CD105, CD90 MSC positive markers and negative CD45 MSC negative markers. Adipogenic, chondrogenic as well as osteogenic experiments revealed that NCMCs possess tri lineage differentiation ability.

4. The NCMCs hydrogel complex cultured in 3D in vitro was stained with living / dead cells and cartilage-specific staining during the differentiation into cartilage. The results showed that in the cellular hydrogel complex, the cell viability was higher, the cell mass formed and the volume increased after 7 days and 21 days of culture. When cultured in vitro for 56 days, the total number of cells in the hydrogel decreased. Alcian blue, fuchsin O and toluidine blue staining showed the formation of cartilage-like cells and matrix.

Conclusions

1. PSHi002-A can be successfully prepared from a small number of peripheral blood monocytes of the patient, and the results show that it meets the preparation standard. PSHi002-A inherits the characteristics of gene mutation in this patient and can obtain a stable and "young" source of cell samples for disease genetic detection. PSHi002-A is qualified to be the "starting point" of the in vitro cartilage development model of this TCS patient.

2. PSHi002-A can differentiate into NCLCs expressing NCCs markers including cranial NCCs marker TWIST1 gene. After screening and culturing the prepared NCLCs, the NCLCs with high positive expression of NCCs markers could be obtained.

3. NCLCs can continue to differentiate into NCMCs with the ability of adipogenesis, chondrogenesis and osteogenesis. In addition, patient-derived NCMCs hydrogel complexes have the ability to differentiate into cartilage in 3D environment in vitro.