从诱导多能干细胞中提取人肾足细胞:从干细胞定向分化成熟肾足细胞的程序

成熟细胞可以被重新编程以恢复其干细胞样未分化和增殖状态。这些重编程的细胞称为诱导多能干细胞或 IPSC。要将多能细胞分化为参与过滤肾脏废物的足细胞特化细胞，首先在中胚层诱导培养基中取人IPSCs悬浮液。

将该悬浮液转移到基质涂层培养板上并孵育。基底基质有助于 IPSC 在基部的粘附。随后，培养基中存在的特定因子诱导 IPSC 分化为中胚层细胞，中胚层细胞是足细胞的前体细胞。吸出用过的介质。

所需体积的中间中胚层诱导培养基补充板并孵育较长时间。这一步导致中胚层细胞分化为未成熟足细胞。用胰蛋白酶溶液替换中间中胚层诱导培养基，以将贴壁细胞从板上解离。

接下来，离心以沉淀细胞。弃去含有胰蛋白酶溶液和基质片段的上清液。将沉淀重悬于足细胞诱导培养基中。将细胞悬液转移到新鲜包被的培养板中。孵育以允许中间细胞发育出几个脚状突起，从而形成成熟的足细胞。

计数后，将细胞重悬至每毫升中胚层诱导培养基浓度的1倍10至第5个细胞，并从基底膜基质2包被板中吸出细胞外基质溶液。用温培养基冲洗板两次，然后将人诱导的多能干细胞悬液与轻柔的移液混合。

1 毫升细胞添加到基底膜基质 2 包被 12 孔板的每个孔中，并轻轻摇动板以使细胞分布更均匀。然后，将板放入细胞培养箱中。在分化的第2至15天，用每孔1毫升中间中胚层诱导培养基替换中胚层诱导培养基。

如果观察到大量细胞生长和营养物质的快速耗尽，如培养基变黄所示，则中间中胚层分化培养基的体积可以增加到每孔1.3毫升。在培养的第16天，用温培养基冲洗中间中胚层细胞，并将细胞与每孔500微升0.05%胰蛋白酶-EDTA在37摄氏度下孵育3分钟。

当细胞开始解离时，用细胞提升器刮擦细胞，并通过移液轻轻混合细胞。用每孔约2毫升胰蛋白酶中和溶液停止反应，并将细胞转移到50毫升锥形管中。用培养基使体积达到50毫升，并通过离心收集细胞。

每毫升培养基浓度的1倍10至第五个细胞重悬沉淀和足细胞诱导培养基，并将细胞添加到基底膜基质2包被板中。然后，轻轻摇动板以帮助更均匀地分布细胞，并将细胞放入培养箱中长达 5 天。

• Induced Pluripotent Stem Cells (IPSCs) - Reprogrammed mature cells with a proliferative, undifferentiated state similar to stem cells.
• Podocytes - Specialized cells involved in the filtration of waste from kidneys.
• Mesoderm Cells - Precursor cells for podocytes that are derived from IPSCs.
• Mature Podocytes - Fully developed podocytes, characterized by several foot-like projections.
• Directed Differentiation - A process of driving the development of undifferentiated cells into a specific cell type.

• Introduce Induced Pluripotent Stem Cells (IPSCs) - Cells which can be reprogrammed to regain a stem cell-like state (e.g., pluripotent).
• Key Concepts - Summarization of the steps from IPSCs to mature podocytes (e.g., directed differentiation of IPSCs, kidney mesoderm).
• Underlying Mechanisms - This involves various elements, such as media induction, cell incubation, and resuspension (e.g., deriving).
• Connect to Experiment - Understanding of how this process is valuable in studying kidney functions (e.g., podocytes kidney, normal human kidney podocytes).

• What are Induced Pluripotent Stem Cells (IPSCs) and how are they reprogrammed?
• What are podocytes and how do they function in the kidney?
• What is the process of deriving podocytes from mesoderm cells?

• Practical Applications - Generation of podocytes could be used in testing pharmaceutical effects on kidneys (e.g., kidney podocytes).
• Industry Impact - The research may offer new treatments for kidney-related diseases (e.g., human podocytes).
• Societal Importance - A better understanding of the IPSCs may revolutionize regenerative medicine (e.g., pluripotent).
• Link to Scientific Advancements - The evolution of technology that could manipulate cell development (e.g., directed differentiation of ipsc).