干细胞生物学概论

JoVE 科学教育
发育生物学
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JoVE 科学教育 发育生物学
An Introduction to Stem Cell Biology

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11:19 min
April 30, 2023

概述

细胞可以分化成多种类型的细胞,称为干细胞,今天都是科学的中心的最令人兴奋领域之一。干细胞生物学家正在试图理解基本的机制,规范如何这些细胞的功能。这些研究人员感兴趣利用非凡潜力的干细胞治疗人类疾病。

在这里,朱庇特介绍干细胞生物学的迷人世界。我们开始与时间线的里程碑研究,从第一个实验证据为造血干细胞在 20 世纪 60 年代,到更近期突破喜欢诱导多功能干细胞。介绍了关于干细胞生物学其次,关键的问题,例如: 这些细胞如何保持其独特能力进行自我更新?这被其次的一些突出的方法,用来回答这些问题进行了讨论。最后,介绍了几个实验来证明干细胞在再生医学中的使用。

Procedure

顾名思义,干细胞是从许多不同的细胞类型”干。”前体他们的特点是他们的效力或从所有三个胚层,以及他们的可再生性或能力来生成更多的干细胞分化的能力。希望以此推进发育生物学和再生医学领域,干细胞研究人员正致力于了解这些独特的细胞是如何完成如此大的壮举。

这部影片讲述了在干细胞生物学,问科学家在这一领域的关键问题,突出的方法,干细胞研究人员,应用程序使用的干细胞研究领域的重大发现。

现在,我们已经介绍了干细胞概念,让我们潜入干细胞研究的丰富历史。

在 20 世纪 60 年代,博士欧内斯特 · 麦卡洛克和詹姆斯耕发现一些存在的成年哺乳动物骨髓中的造血干细胞第一次决定性的证据。这些细胞有自我更新的能力,而且还多能干,意味着它们可以分化成多种不同的但有限,细胞即血液和免疫系统的细胞类型。

1988 年,美国斯坦福大学和同事完善纯化小鼠骨髓造血干细胞的一种方法。

1981 年,教授盖尔 · 马丁创造了术语”胚胎干细胞”。不同于造血干细胞,胚胎干细胞是多能干细胞,具有分化成人体的所有细胞类型的能力。她和科学家马丁 · 埃文斯和马修 · 考夫曼同时,但分开,开发方法提取小鼠囊胚内细胞团和培养他们在体外作为干细胞。

1998 年,十多年后的小鼠胚胎干细胞,分离博士詹姆斯 · 汤姆森成功建立第一个人类胚胎干细胞线。

2006 年,一项重大突破发生随着诱导多能干细胞,作为由博士 Shinya Yamanaka 设计。山中伸弥约翰格登的工作的基础上由使用逆转录病毒诱导表达的转录因子,现在被称为”山中伸弥因素。”一小套开发改编分化细胞到胚胎状态的方法 由此产生的细胞被命名为”诱导多能性干细胞”或”诱导多能干细胞。”这些实验被认为从根本上重要,山中伸弥和格登了 2012 年授予诺贝尔奖。

目前,我们现在在多个组织中有几种人类疾病和再生平台 iPSC 型号。

今天,干细胞研究是由几个首要问题驱动的。

其中最重要的这些问题是: 干细胞如何保持多向分化潜能和可再生性?有两个相关的特性赋予这些属性的干细胞。首先是特定基因表达的细胞和自我更新的必要条件。第二是干细胞向影响这些基因表达的调节因子的响应能力。

下一个问题是: 如何是干细胞定向分化的吗?如干细胞发育成成熟的细胞,激活的具体分化通路诱导基因表达,干细胞基因关闭和开启组织特异性基因,导致细胞功能和形态的专业化不断提高的变化。

最后,让我们谈谈驾驶干细胞研究经费的主要问题: 可以用干细胞治疗疾病吗?再生医学解决这一问题在两个方面: 1) 由再生器官在实验室里,和 2) 通过提供干细胞通过移植治疗组织退行性变。

现在,我们已经介绍了一些关于干细胞生物学,咱们过去的一些突出的方法用于解决他们的关键问题。

微阵列技术可以用来发现哪些基因赋予效价和可再生性干细胞。在这种技术,总 RNA 是从一群细胞,作为当前基因表达的快照隔离。在一系列的步骤,此 mRNA 是转换成荧光标记探针和杂交到芯片上包含的整个人类基因组的转录。扫描此芯片提供相关基因表达谱读出。正如你所看到的干细胞表达一组特定的基因不同于一个分化的细胞。

另一种测定多向分化潜能基因涉及 Oct4 GFP 检测系统。Oct4 是需要自我更新,分化过程中是快速下调。因此,其表达式作为一个可靠指标的细胞。在这个实验中,细胞表达绿色荧光蛋白 Oct4 基因启动子控制下。然后可以通过实验操纵这些细胞,并在绿色荧光蛋白表达变化的分析,找出新的基因或调节自我更新的可溶性因子。

为了研究干细胞在体外,我们必须首先了解如何培养他们。干细胞需要一个特殊的微环境,维持细胞。这可以通过干细胞饲养层细胞,如小鼠胚胎成纤维细胞或小鼠细胞联合培养。小鼠分泌必要多分化潜能性和自我更新因素的复杂的混合物。

有时,它是需要有无饲养层培养干细胞。保持无饲养层细胞系的主要方法是补充细胞培养基与股票试剂的增长和抑制因素。

使用几种方法,完成了干细胞体外分化。差异表达基因是最终负责专业化的细胞。在两步方法你看到这里,才有进一步的神经元的命运引物体外培养的小鼠胚胎干细胞分化与运动神经元诱导培养基。这些因素激活基因表达,从而导致形态和蛋白质组学变化特征的运动神经元的具体途径。

传统的体外分化的一个主要缺点是平板限制 3D 细胞生长。垂悬滴法和微胶囊的方法绕过这些问题。中悬滴技术,干细胞悬液的小水滴是镀上的皮氏培养皿盖子,颠倒了体外培养至窗体集料的干细胞被称为胚状体机构。

在微胶囊化方法中,拌干细胞生物相容性的半透膜称为藻酸盐,和作为珠存入细胞培养板。这两种方法允许进一步分化为专门的细胞,如多巴胺能神经元和心肌细胞。

知道如何直接分化是利用干细胞在再生医学中的重要一步。干细胞移植疗法的目的是治疗和治疗退行性疾病的干细胞修复受损组织。在这个实验中,从病人体细胞被重新编码成 iPS 细胞通过 lentivral 感染的山中伸弥因素。从他们多能干的状态,细胞分化成特定细胞类型并返回到主机,修复受损的组织。

现在,你知道一些用来研究干细胞的方法,让我们看看如何将这些方法应用在具体实验中。

在这个实验中使用的多发性硬化症的小鼠模型,神经干细胞静脉注入受影响小鼠。治疗小鼠脑片收集和评估成功移植在显微镜下成像。来自捐助神经元前体细胞细胞跟踪使用报告基因 LacZ。正如你所看到的供者干细胞数目有区分和纳入的病鼠中枢神经系统。

不是每个疾病可以通过系统性注射治疗。软骨损伤,例如,需要专门的脚手架周围重建。在这个实验中,骨髓间充质干细胞和凝血因子的混合物养殖在一起形成血栓。血凝块然后是放入一只兔子的损伤的膝盖软骨,并允许集成。通过执行此过程,重塑的膝关节软骨到一个光滑和功能的联合可以观察到。

有时,干细胞研究人员和组织工程师组队来重建整个器官。在这个实验中,灵长目动物肺清洗一次以 decellularize 器官,留下的只有非蜂窝结构的组件。这个”鬼”肺被转移到一种生物反应器,在那里它种子与血管和上皮干细胞。为了进一步模拟自然肺经验的行为与压力,生物反应器循环介质、 保持压力和气体水平,膨胀与肺部。

你刚看了朱庇特的干细胞生物学概述。回顾一下,在这个视频我们讨论了干细胞及其历史、 维护、 分化和交付方法和干细胞的应用程序。谢谢观赏 !

成績單

As their name implies, stem cells are the precursors from which many different cell types “stem.” They are characterized by their potency, or ability to differentiate into cells from all three germ layers, as well as their renewability, or capacity to generate more stem cells. In hopes of advancing the fields of developmental biology and regenerative medicine, stem cell researchers are working to understand how these unique cells accomplish such a major feat.

This video covers major discoveries in the field of stem cell biology, key questions asked by scientists in this field, prominent methods used by stem cell researchers, and applications of stem cell research.

Now that we’ve introduced the stem cell concept, let’s dive into the rich history of stem cell research.

In the 1960s, Drs. Ernest McCulloch and James Till discovered some of the first definitive evidence for the existence of hematopoietic stem cells in the bone marrow of adult mammals. These cells have the ability to self-renew and are multipotent, meaning that they can differentiate into multiple, but limited, types of cells-namely the cells of the blood and immune systems.

In 1988, Irving Weissman and colleagues perfected a method for purification of hematopoietic stem cells from mouse bone marrow.

In 1981, Professor Gail Martin coined the term “embryonic stem cell.” Unlike hematopoietic stem cells, embryonic stem cells are pluripotent, having the ability to differentiate into all cell types of the body. She and scientists Martin Evans and Matthew Kaufman, simultaneously, but separately, developed methods to extract the inner cell mass from mouse blastocysts and culture them in vitro as stem cells.

In 1998, over ten years after the isolation of mouse embryonic stem cells, Dr. James Thomson successfully established the first human embryonic stem cell lines.

In 2006, a major breakthrough occurred with the advent of induced pluripotent stem cells, as devised by Dr. Shinya Yamanaka. Building on the work of John Gurdon, Yamanaka developed a method to reprogram differentiated cells to a pluripotent state by using retrovirus to induce the expression of a small set of transcription factors now known as the “Yamanaka factors.” The resultant cells were named “induced pluripotent stem cells,” or “iPSCs.” These experiments were considered so fundamentally important that Yamanaka and Gurdon were awarded the Nobel Prize in 2012.

At present, we now have iPSC models of several human diseases, and regeneration platforms in multiple tissues.

Today, stem cell research is driven by several overarching questions.

One of the most important of these questions is: how do stem cells maintain pluripotency and renewability? There are two related characteristics of stem cells that confer these properties. First is the expression of specific genes essential for stemness and self-renewal. The second is the responsiveness of stem cells to regulatory factors that affect the expression of these genes.

The next logical question is: how is the differentiation of stem cells directed? As a stem cell develops into a mature cell, activation of specific differentiation pathways induces changes in gene expression, turning off stem cell genes and turning on tissue-specific genes, which results in increasing specialization of cell function and morphology.

Finally, let’s address the main question driving stem cell research funding: can stem cells be used to treat disease? Regenerative medicine is tackling this question in two ways: 1) by regrowing organs in the lab, and 2) by delivering stem cells via transplantation to treat tissue degeneration.

Now that we’ve presented some of the key questions concerning stem cell biology, let’s go over some of the prominent methods used to address them.

Microarray technology can be employed to discover which genes confer potency and renewability in stem cells. In this technique, total RNA is isolated from a population of cells, which acts as a snapshot of current gene expression. In a series of steps, this mRNA is converted to a fluorescently labeled probe and hybridized to a chip containing transcripts of the entire human genome. Scanning this chip provides a readout of relative gene expression profiles. As you can see, a stem cell expresses a specific set of genes that differs from a differentiated cell.

Another assay for pluripotency genes involves the Oct4-GFP detection system. Oct4 is required for self-renewal, and is quickly down-regulated during differentiation. Therefore, its expression acts as a reliable indicator of “stemness.” In this experiment, cells express green fluorescent protein under the control of the Oct4 promoter. These cells can then be experimentally manipulated, and changes in GFP expression are analyzed to identify new genes or soluble factors that modulate self-renewal.

In order to study stem cells in vitro, we must first understand how to culture them. Stem cells require a particular microenvironment to maintain stemness. This can be achieved by co-culturing stem cells with feeder cells, such as mouse embryonic fibroblasts, or MEFs. MEFs secrete a complex mixture of necessary pluripotency and self-renewal factors.

At times, it is desirable to have feeder-free cultures of stem cells. The main method to maintain feeder-free cell lines is to supplement cell culture media with stock reagents of growth and inhibitory factors.

Differentiating stem cells in vitro is accomplished using several methods. Differential gene expression is ultimately responsible for specialization of cells. In the two-step method you see here, cultured mouse embryonic stem cells are primed for a neuronal fate before being further differentiated with motor neuron induction medium. These factors activate specific pathways of gene expression, resulting in morphological and proteomic changes characteristic of motor neurons.

One major disadvantage of traditional in vitro differentiation is that flat plates restrict the 3D growth of cells. The hanging drop method and microcapsule methods circumvent these issues. In the hanging drop technique, small drops of stem cell suspensions are plated on the lid of a petri dish and cultured upside down to form aggregates of stem cells known as embryoid bodies.

In microencapsulation methods, stem cells are mixed with a biocompatible semipermeable membrane called alginate, and deposited as beads into cell culture plates. Both methods allow for further differentiation into specialized cells, such as dopaminergic neurons and cardiomyocytes.

Knowing how to direct differentiation is a major step towards using stem cells in regenerative medicine. Stem cell transplantation therapy aims to treat and cure degenerative diseases by repairing damaged tissues with stem cells. In this experiment, somatic cells from patients are reprogrammed into iPS cells via lentivral infection of the Yamanaka factors. From their pluripotent state, cells are differentiated into specific cell types and returned to the host to repair damaged tissue.

Now that you know some of the methods used to investigate stem cells, let’s take a look at how these methods are applied in specific experiments.

In this experiment using a mouse model of multiple sclerosis, neural stem cells are injected intravenously into affected mice. Brain slices of treated mice are collected and imaged under a microscope to assess success of the transplantation. Cells derived from donor neuronal precursor cells are tracked using the reporter gene LacZ. As you can see, a number of donor stem cells have differentiated and integrated into the central nervous system of diseased mice.

Not every ailment can be treated by systemic injections. Cartilage injuries, for example, require a specialized scaffolding to rebuild around. In this experiment, a mixture of mesenchymal stem cells and coagulation factors are cultured together to form a clot. The clot is then placed into the damaged knee cartilage of a rabbit and allowed to integrate. Following this procedure, remodeling of the knee cartilage to a smooth and functional joint can be observed.

Sometimes, stem cell researchers and tissue engineers team up to rebuild entire organs. In this experiment, primate lungs are washed to decellularize the organ, leaving behind only non-cellular structural components. This “ghost” lung is then transferred to a bioreactor, where it is seeded with vascular and epithelial stem cells. To further mimic the pressure and behavior that a natural lung experiences, the bioreactor circulates media, maintains pressure and gas levels, and inflates the lungs.

You’ve just watched JoVE’s stem cell biology overview. To recap, in this video we have discussed stem cells and their history, maintenance, differentiation and delivery methods, and stem cell applications. Thanks for watching!

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