研究背景和目的：

    肥大细胞（Mast cells, MC）和巨噬细胞（Macrophage, MΦ）是非常重要的免疫细胞，在机体固有免疫反应中具有举足轻重的作用。MC和MΦ具有极大的异质性。在人类MΦ既有分布于血液中以单核形式存在的状态，也有在多种组织和器官中分布的组织驻留型，并根据分布的形态和功能具有不同命名；而人类MC根据颗粒中含有蛋白酶种类不同，分为MC_T和MC_TC两类，前者与小鼠黏膜MC相似，后者与小鼠组织相关MC类似。MΦ和MC表面和内体分布多种Toll样受体（TLRs，一种机体内重要的模式识别受体），可有效识别病原体相关外源性危险信号（PAMPs）和自身细胞所释放的内源性危险信号（DAMPs），在机体防御中发挥着重要的作用。

    在个体发生的早期阶段，血细胞起源在顺序上有着时空分布的不同特征。所有血细胞均来源于中胚层，最早的造血细胞起源于卵黄囊；随后进入胚内的主动脉-性腺-中肾区域（AGM区），在此可产生最初的能够确认的造血干细胞；最后进入胎肝造血，在此含有大量可移植的造血干细胞。依据人体造血发生的时空顺序及不同特征，可将其分为原始造血和成体造血。在成体造血，所有功能型血细胞均来源于骨髓中的造血干细胞（Hematopoietic stem cells, HSCs）。然而，对小鼠胚胎造血发育的研究显示，起源于卵黄囊的原始造血以及红系/髓系祖细胞（EMP）都可产生MΦ。另外，在小鼠胎肝发育过程中发现MC前体高度集中于卵黄囊和胎肝血中，提示MΦ和MC存在一个较强的早期胚性发育阶段。研究发现，产生于早期胚性发育阶段的这两种固有免疫细胞具有组织偏向特性。由于受伦理道德及法律的限制，在人体中进行胚胎造血发育的研究存在诸多困难，导致对人类早期胚胎造血发生认识的缺失。

对造血细胞发生学的研究主要基于小鼠等动物模型。但是动物体内的研究数据并不能完全指示人体内的发育过程。随着人胚胎干细胞（human embryonic stem cells, hESCs）的成功建系以及人诱导性多能干细胞（human induced pluripotent stem cells, hiPSCs）的体外重编程获得，为研究人类早期发育机制提供了有效的手段。hESCs和hiPSCs统称为人多能干细胞（human pluripotent stem cells, hPSCs），具有自我更新、无限增殖及分化形成完整个体的能力，为研究跨胚层细胞、组织以及所有成体细胞的发育，提供了有效的手段。已有大量关于从hPSCs体外成功分化出三个胚层来源成熟细胞的报道。基于hPSCs向造血细胞诱导分化的研究，可很大程度上重现早期人类造血发生过程，有助于详尽地解析人类胚胎期造血的发育模式。

为了研究人类早期发育过程中MC和MΦ的发育情况，从而更好地模拟体内早期发育，需要在体外建立一种高效地趋于自然发育过程的培养体系。通过与成体HSCs来源固有免疫细胞比较，我们研究了早期造血发育产生固有免疫细胞的功能及识别能力。在此，我们以人固有免疫细胞初期发育作为研究方向，重点探索hPSCs体外诱导产生MC和MΦ初期发育的起源及其功能。

研究方法:

    基于hPSCs/AGM-S3共培养体系，建立固有免疫细胞体外培养方法。以MC和MΦ为主，分别检测两种固有免疫细胞的形态、表型特征以及功能，并探索两种细胞的起源。具体操作方法如下：

 以小鼠主动脉-性腺-中肾（Aorta-Gonad-Mesonephros, AGM）来源基质细胞系（AGM-S3），与hPSCs体外共培养，诱导产生大量造血干/祖细胞，进一步悬浮培养定向产生MΦ和MC。培养方法分为三步：

将hPSCs细胞接种到AGM-S3上进行共培养，诱导造血分化发生，监测不同时间点克隆形态及表型分子表达变化，

经悬浮培养一周，扩增造血干/祖细胞，

更换培养基进行MC或MΦ定向分化。

 观察两种固有免疫细胞形态及表型特征

1）MC形态学特征：麦格氏-吉姆萨（May-Grunwald Giemsa, MGG）、酸性甲苯胺蓝（Acidic toluidine blue）以及阿尔新蓝（Alcian blue）化学染色观察细胞形态；电镜观察MC内颗粒形态；免疫荧光染色观察MC中Carboxypeptidase A、Cathepsin-G、Tryptase和Chymase蛋白酶的表达情况，多色流式检测技术分析MC表型分子表达谱系。

2）MΦ形态学特征：MGG染色观察三种来源MΦ形态；多色流式检测技术分析三种来源MΦ表型分子表达谱系。

3. 检测两种固有免疫细胞功能

1）MC功能检测：酶联免疫吸附法检测IgE、Substance P和Compound48/80刺激下，MC上清及细胞沉淀中组胺的含量；RT-qPCR方法检测TLRs表达。

2） MΦ功能检测：利用RT-qPCR方法检测不同极化条件下，炎症因子及TLRs表达；通过低密度脂蛋白（LDL）吞噬实验检测MΦ吞噬能力。

4. 寻找MC的祖细胞：利用流式分选技术（Fluorescence Activated Cell Sorting, FACS）分选目标细胞群，确定MC祖细胞

结果： 

建立高效的hPSCs/AGM-S3共培养造血细胞诱导分化体系，可产生大量高纯度MC和初期MΦ。

共培养14天再向MC定向培养10天，体系中发现C-KIT⁺, CD45⁺, Tryptase⁺和Chymase⁺的MC，40天双阳比例高达98%。

共培养3天再向MΦ定向培养14天，可产生初期MΦ。

产生的两种固有免疫细胞具有组织偏向型

1）化学染色，免疫荧光染色，电镜观察以及IgE、Substance P和Compound48/80刺激下，MC都可释放组胺，确定我们的培养体系产生的MC为MC_TC类型。

2）初期产生MΦ在IL-4刺激可有效极化成M2型

产生的两种固有免疫细胞与成体HSCs来源固有免疫细胞不同

1）以外周血单个核细胞作为对照，与脐带血CD34⁺细胞来源的MC相比，hESCs/AGM-S3共培养产生的MC高表达TLR2，TLR4和TLR9。

2）与脐带血CD34⁺细胞来源MΦ以及共培养14天来源MΦ相比，共培养3天来源MΦ表型分子具有明显不同的表达水平。IL-4刺激共培养3天来源MΦ中TLR1~TLR8基因表达水平明显高于前两种来源MΦ。

4. 产生的两种固有免疫细胞诞生于成体造血干/祖细胞(CD34⁺CD45⁺)之前

1）流式分选共培养第8天CD34⁺C-KIT^-和CD34⁺C-KIT⁺两群细胞，向MC定向培养14天，免疫荧光染色显示两亚群产生子代细胞Tryptase和Chymase双阳百分比都大于85%。

2）共培养第3天产生的MΦ，诞生于成体造血干/祖细胞之前。

结论：

1. hPSCs/AGM-S3造血共培养体系可诱导产生MC和MΦ等固有免疫细胞，并且在成体造血干/祖细胞（CD34⁺CD45⁺细胞）诞生之前，可产生一群MC和MΦ。

2. 这群MC和MΦ具有自己独特的发育途径。

3. 这些初期产生的固有免疫细胞偏向于组织特性，高表达TLRs，说明分布于组织中的MC和MΦ具有更强的免疫识别能力，在固有免疫反应中发挥着重要的作用。

Background and Objective:

Mast cells (MC) and macrophages (MΦ) are very important innate immunity cells, which play key roles in immunity response and also have great heterogeneity. MΦ exist in the form of mononuclear/MΦ both in the blood and variety of tissues with different names according to the morphology and their locations. The MC_TC and MC_T types of human MC were initially recognized as the basis of the different protease compositions of their secretory granules, with tryptase, chymase, Carboxypeptidase A and Cathepsin G in the former and only tryptase in the latter. MC_T are the predominant type found in the lungs, bronchial airway basement membrane and adjacent sub-mucosal glands parts in free form, similar with mouse mucosal MC. While MC_TC are the predominant type found in the skin around the vascular connective tissue in the sub-mucosa, and mouse tissue mast cell related similar. A variety of toll-like receptors (TLRs) are found on the surface and body distribution of MC and MΦ, as the one of important pattern recognition receptors in the body, TLRs can effectively identify the pathogen related exogenous danger signals and release endogenous danger signals, playing an important role in the body's defenses.

In the early development of an individual, the origin of the blood cells is characterized by the chronological order. All the blood cells are derived from mesoderm, and the earliest hematopoietic cells are found in the yolk sac. Later it occurred in the Aorta-Gonad- Mesonephros (AGM) region, where the original hematopoietic stem cells (HSCs) are found. Finally, HSCs seed in the fetal liver, where the HSCs can be transplanted. Based on the chronological order and different characteristic of human hematopoiesis, it is divided into primitive hematopoiesis and definitive hematopoiesis. The study of adult hematopoiesis has confirmed that all functional blood cells are derived from bone marrow hematopoietic stem cells (HSCs). However, the study of hematopoiesis during mouse embryonic development showed that there are three waves. Both the primitive hematopoiesis and erythromyeloid progenitors (EMPs) originated from yolk sac can produce MΦ. In addition, during the development of mouse fetal liver, it was found that precursors of MC were highly concentrated in yolk sac and fetal liver blood. The data above showed that there is a strong early embryonic development stage of MΦand MC, and the study found that two kinds of cells produced in this stage have tissue bias. However, due to the restrictions of ethics and law, it is difficult to study the development of embryonic hematopoiesis directly in human body.

As the lack of a suitable model to investigate early human hematopoiesis, previous studies have been based on animal models, such as mice. However, the data from animal studies do not fully indicate the development of the human body. Successful establishment of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) provided an accessible model for studying early embryonic development of human. Human pluripotent stem cells (hPSCs), including hESCs and hiPSCs, have the ability of self-renewal, proliferation and differentiation into all types cell. It is widely reported that hPSCs can be differentiated into all mature cells of three germ layers. Based on the research of hPSCs-drevied blood cells, the process of early human hematopoiesis can be largely reproduced and provide a good model to the scientific community for more detailed analyzing human embryonic hematopoiesis.

In order to study the early development of human MΦ and MC, we need to establish a highly efficient culture system in vitro, to better mimic the in vivo developmental process. After compared the cells derived from the adult hematopoietic stem cells, we study that the function and reorganization of the innate immune cells derived from the early development. Here, we take the initial development of innate immune cells as the research direction, and study the origin and function of macrophages and MC differentiated from hPSCs in vitro.

Methods: 

Based on the co-culture system of hPSCs/AGM-S3, an innate cells cultured method was established. In the main of MC and MΦ, we detected the morphology, phenotype, function as well as explored the origins of the two types cells. The operation methods are as follow:

Use mouse aorta gonad mesonephros (Aorta-Gonad-Mesonephros, AGM) cells as stromal cell, co-culture with hPSCs in vitro, induced a large amount of hematopoietic stem/progenitor cells, then using directional suspension culture method for MC and MΦ. The process can be divided into three steps:

1) Inoculate hPSCs clones into stromal cells, induce hematopoietic and detect the phenotypes of the clones and the expression of surface markers at different time points.

2) Suspension culture for 7 days, amplificated the hematopoietic stem/progenitor cells,

3) Changing medium for MC or MΦ directional differentiation.

2. Observe morphology and phenotype characters of MC and MΦ

1) MC: Use MGG, toluidine blue and alcian blue method to observe the morphology of MC, observe the granules of MC by transmission electron microscope, Immunofluorescence (IF) staining analyze the expressing of Tryptase and Chymase in MC, FACS analysis of surface marker on MC.

2) MΦ: The three conditions are (a) cord blood mononuclear cells CD34⁺ derived macrophages, (b) co-culture D3 cells derived macrophages and (c) co-culture day14 cells, observed the morphology with MGG, detected the expression of surface markers on the three conditions macrophages with multicolor flow cytometric analysis technology (FACS).

3. Detecte the function of MC and MΦ

1) Function of MC: Detection of histamine release by Enzyme-linked immune (ELISA) analysis, detected the expression level of TLRs with RT-qPCR.

2) Using RT-qPCR detected the expression of inflammatory and TLRs under different polarization conditions, detecting phagocytosis of macrophages with low-density lipoprotein.

4. Use FACS technology to sort the target cells, find out the progenitor cells of MC.

Results:

Established a high-efficient hPSCs/AGM-S3 co-culture hematopoietic differentiation system, which can produce a large number of MC with high purity and early MΦ

1) Through co-culture 14 days then cultured 10 days, the double positive tryptase and chymase MC are found, the percent is up to 98% on 40 days. The number of C-KIT⁺ Tryptase⁺ MC is 200 times of undifferentiated H1 cells.

2) By co-culture 3 days then cultured 14 days, early MΦ were produced.

Both MC and MΦ have an organized bias

Immunological staining, IF and electron microscopy, IgE receptor antibody, substrate P, and complex 48/80 can stimulate MC release histamine, based on the data, and confirmed the MC in our culture system is MC_TC type.

Early MΦ can be polarized into M2 type by IL-4.

MC_TC and early MΦ are different with the cells generated from adult HSCs

Compared with the MC derived from peripheral blood and cord blood CD34⁺ cells, hPSCs/AGM-S3-derived MC have high expression of TLR2, TLR4 and TLR9.

The expression of surface markers of the macrophages produced from the three different culture conditions are significantly different from the others. The expression levels of TLR1~TLR8 were significantly higher in M2 macrophages derived from co-culture day 3 cells.

MC_TC and MΦ were produced before adult HSCs（CD34⁺CD45⁺）

Sorted D8 cells by FACS analysis, the daughter cells from CD34⁺C-KIT^- and CD34⁺C-KIT⁺ cells fraction, having a high positive expression of tryptase and chymase for 14 days suspension culture.

There is no double positive CD34 and CD45 cells in co-culture Day3, so the early MΦ was born before adult HSCs.

Conclusion:

1. The hPSCs/AGM-S3 co-culture system can induce innate immune cells, such as MC and macrophages, and those cells are produced earlier than the adult hematopoietic stem cells, in our system it means CD34⁺CD45⁺ cells.

2. These early innate immune cells have unique development pathway, and independent from HSCs.

3. These early innate cells tend to tissue characteristics and have high expression of TLRs，showing the MC_TC and MΦ in tissue have higher ability of recognition, and may play an important role in innate immunity response.