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1. Human iPSC Differentiation to Macrophages
NOTE: A schematic summary of the macrophage differentiation protocol is depicted in Figure 1.
<ol>
	<li>Preparing cell differentiation growth factors and other reagents
	<ol>
		<li>Prepare human embryonic stem cell-serum-free media (hESC-SFM; see Table of Materials) by supplementing Dulbecco&#39;s modified Eagle medium-F12 (DMEM/F12) with hESC supplement, 1.8% w/v bovine serum albumin (BSA), and 0.1 mM 2-mercaptoethanol.</li>
		<li>Prepare 0.1% w/v solution of porcine gelatin by dissolving the gelatin into sterile water. Gelatin solution can be stored at 4 &#176;C for up to 2 years.</li>
		<li>Prepare human bone morphogenetic protein (BMP4) stock solution (25 &#956;g/mL) by dissolving BMP4 into a 4 mM hydrogen chloride (HCl)-0.2% BSA PBS solution. Distribute the stock solution as 50 &#956;L aliquots in cryotubes. Stock solutions can be stored at -20 &#176;C for up to 1 year. Once thawed, stock BMP4 can be stored at 4 &#176;C for 5 days.</li>
		<li>Prepare human vascular endothelial growth factor (VEGF) stock solution (100 &#956;g/mL) by dissolving VEGF into a 0.2% BSA PBS solution. Distribute the stock solution as 10 &#956;L aliquots in cryotubes. Stock solutions can be stored at -20 &#176;C for up to 1 year. Once thawed, stock VEGF can be stored at 4 &#176;C for 7 days.</li>
		<li>Prepare human stem cell factor (SCF) stock solution (100 &#956;g/mL) by dissolving SCF into a 0.2% BSA PBS solution. Distribute the stock solution as 5 &#956;L aliquots in cryotubes. Stock solutions can be stored at -20 &#176;C for up to 1 year. Once thawed, stock SCF can be stored at 4 &#176;C for 10 days.</li>
		<li>Prepare human interleukin-3 (IL3) stock solution (10 &#956;g/mL) by dissolving IL3 into a 0.2% BSA PBS solution. Distribute the stock solution as 500 &#956;L aliquots in cryotubes. Stock solutions can be stored at -20 &#176;C for up to 2 years. Once thawed, stock IL3 can be stored at 4 &#176;C for 15 days.</li>
		<li>Prepare human macrophage colony-stimulating factor (CSF1) stock solution (10 &#956;g/mL) by dissolving CSF1 into a 0.2% BSA PBS solution. Distribute the stock solution as 1 mL aliquots in cryotubes. Stock solutions can be stored at -20 &#176;C for up to 2 years. Once thawed, stock CSF1 can be stored at 4 &#176;C for 15 days.</li>
	</ol>
	</li>
	<li>Stage 1: Generation of embryoid bodies (day 0&#8211;day 3)
	<ol>
		<li>On day 0, add 2.25 mL of Stage 1 media (hESC-SFM supplemented with 50 ng/mL BMP4, 50 ng/mL VEGF, and 20 ng/mL SCF) into two wells of an ultralow attachment 6-well plates.</li>
		<li>Replace maintenance media of one 80% confluent well of iPSCs in a 6-well plate with 1.5 mL of Stage 1 media.</li>
		<li>Cut colonies using the cell passaging tool and transfer cut colonies with a pipette into the two wells of an ultralow attachment 6-well plate (see Table of Materials).</li>
		<li>On day 2, bring cytokines to a final concentration of 50 ng/mL BMP4, 50 ng/mL VEGF, and 20 ng/mL SCF using 0.5 mL of hESC-SFM media. 
		NOTE: IPSC colonies will become EBs (Figure 2A).</li>
	</ol>
	</li>
	<li>Stage 2: Emergence of hematopoietic cells in suspension
	<ol>
		<li>On day 4, coat 4 wells of a 6-well tissue culture plate with 0.1% w/v gelatin and incubate for at least 10 min.</li>
		<li>Remove gelatin and add 2.5 mL of Stage 2 media (X-VIVO15 supplemented with 100 ng/mL CSF1, 25 ng/mL IL-3, 2 mM glutamax, 1% penicillin-streptomycin, and 0.055 mM 2-mercaptoethanol).</li>
		<li>Collect formed EBs into a 50 mL centrifuge tube and allow them to settle at the bottom of the tube by gravity. Carefully aspirate the media.</li>
		<li>Resuspend EBs in 2 mL of Stage 2 media.</li>
		<li>Transfer 10&#8211;15 EBs (no more than 15) to a gelatin-coated well containing 2.5 mL of Stage 2 media.</li>
		<li>Incubate EBs at 37 &#176;C and 5% CO2 air.</li>
		<li>Change media on plated EBs every 3&#8211;4 days for 2&#8211;3 weeks.</li>
		<li>After 2&#8211;3 weeks, the EBs start releasing nonadherent hematopoietic cells into suspension. 
		NOTE: This period of suspension cell release can vary and is cell line dependent. Cells in this suspension can be harvested and matured into macrophages (see Stage 3) (Figure 2A).</li>
	</ol>
	</li>
	<li>Stage 3: Terminal macrophage maturation
	<ol>
		<li>Collect suspension hematopoietic cells and replenish media (Stage 2 media) on the EB plate.</li>
		<li>Centrifuge suspension cells at 200 x g for 3 min.</li>
		<li>Resuspend suspension cells in Stage 3 media (X-VIVO15 supplemented with 100 ng/mL CSF1, 2 mM glutamax, and 1% penicillin-streptomycin).</li>
		<li>Plate collected and spun cells onto untreated plastic 10 cm bacteriological-grade plates (10 mL) or uncoated 6-well tissue culture plates (3 mL) at a 0.2 x 106 cells/mL density.</li>
		<li>Keep cells in Stage 3 media for 9&#8211;11 days, changing media every 5 days. 
		NOTE: Steps 1.4.1&#8211;1.4.5 from Stage 3 can be repeated every 3&#8211;4 days and suspension cells can be harvested from the original EB plate for up to 3 months.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>CellMask Deep Red Plasma Membrane Stain</td><td>Thermofisher</td><td>C10046</td><td>Cryopreservation media</td></tr><tr><td>Cryostor CS10</td><td>Sigma</td><td>C2874</td><td></td></tr><tr><td>CTS CELLstart Substrate</td><td>Invitrogen</td><td>A1014201</td><td>Stem cell substrate</td></tr><tr><td>Porcine Skin Gelatin</td><td>Sigma</td><td>G9136</td><td></td></tr><tr><td>Recombinant Human BMP4 Protein</td><td>R&amp;amp;D</td><td>314-BP-010</td><td></td></tr><tr><td>Recombinant Human IL3</td><td>Preprotech</td><td>0200-03-10</td><td></td></tr><tr><td>Recombinant Human MCSF (carrier-free) 100ug</td><td>Biolegend</td><td>574806</td><td></td></tr><tr><td>Recombinant Human VEGF Protein</td><td>R&amp;amp;D</td><td>293-VE-010</td><td></td></tr><tr><td>SCF (C-Kit Ligand) Recombinant Human Protein</td><td>Thermofisher</td><td>PHC2111</td><td></td></tr><tr><td>StemPro hESC SFM</td><td>Thermofisher</td><td>A1000701</td><td></td></tr><tr><td>StemPro EZPassage Disposable Stem Cell Passaging Tool</td><td>Thermofisher</td><td>23181010</td><td></td></tr><tr><td>Ultralow attachment plates: Cell culture multi-well plate, 6 well, cell star cell repellent surface</td><td>Greiner</td><td>657970</td><td></td></tr><tr><td>2-Mercaptoethanol (50 mM)&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>31350010</td><td></td></tr><tr><td>DPBS, calcium, magnesium (500ml)</td><td>Thermofisher&amp;nbsp;</td><td>14040091</td><td></td></tr><tr><td>GlutaMAX Supplement&amp;nbsp;</td><td>Thermofisher&amp;nbsp;</td><td>35050061</td><td></td></tr></tbody></table>

differentiation of human-induced pluripotent stem cells into macrophages

Source: Lopez-Yrigoyen, M., et al. <a href="https://app.jove.com/v/61038">Production and Characterization of Human Macrophages from Pluripotent Stem Cells.</a> J. Vis. Exp. (2020).
This video describes an in vitro method for generating macrophages from human induced pluripotent stem cells (iPSCs). Macrophage generation from iPSCs occurs in three stages: (i) aggregation of iPSCs in suspension to form three-dimensional embryoid bodies (EBs) that differentiate into mesodermal cells, (ii) formation of macrophage precursor cells from the differentiated EBs, and (iii) differentiation of the macrophage precursor cells into mature, functional macrophages.

<img alt="Figure 1" src="/files/ftp_upload/21516/21516_Figure1.jpg" /> 
Figure 1: Graphic summary of iPSC differentiation to mature functional macrophages. Diagram drawn with Biorender.
<img alt="Figure 2" src="/files/ftp_upload/21516/21516_Figure2.jpg" /> 
Figure 2: iPSC differentiation towards macrophages and iPSC-DM number and morphology. (A) Bright Field images obt...

Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages

Source: Lopez-Yrigoyen, M., et al. Production and Characterization of Human Macrophages from Pluripotent Stem Cells. J. Vis. Exp. (2020).
This video ...

Adult somatic cells can be genetically reprogrammed to obtain induced pluripotent stem cells, iPSCs, that exhibit stem cell-like undifferentiated and proliferative states.&#160;
To differentiate human iPSCs into macrophages &#8212; white blood cells that phagocytose pathogens and cellular debris &#8212; begin with a culture plate with a non-cell adhesive surface.
Add media containing the growth factors bone morphogenetic protein, stem cell factor, and vascular endothelial growth factor. Seed the iPSCs and incubate.
The non-adhesive surface prevents iPSC attachment and maintains the cells in suspension, facilitating aggregation into three-dimensional embryoid bodies, EBs. The growth factors initiate iPSC differentiation within the EBs into mesodermal lineage cells.
Transfer the differentiated EBs to a centrifuge tube. Allow them to settle through gravity. Resuspend the EBs in fresh media containing interleukin-3, IL3, and macrophage colony-stimulating factor, CSF1. Plate the EBs on a gelatin-coated cell culture plate.
Gelatin &#8212; an extracellular matrix protein &#8212; facilitates EB attachment. IL3 stimulates the EBs to differentiate into hematopoietic stem-progenitor cells, giving rise to myeloid cells. CSF1 supports myeloid cell proliferation and their differentiation into macrophage precursors that get released and remain in suspension in the media.
Collect the suspension cells and centrifuge. Resuspend the cells in CSF1-supplemented media.
Plate the cells on uncoated culture plates.
CSF1 induces terminal differentiation of the macrophage precursors into fully-functional, adherent mature macrophages.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Differentiation Techniques

252 次观看. 来源:Lopez-Yrigoyen， M. 等人。多能干细胞中人巨噬细胞的生产和表征。J. Vis. Exp.（2020 年）。 该视频介绍了一种从人诱导多能干细胞 （iPSC） 生成巨噬细胞的体外方法。iPSC 的巨噬细胞生成分为三个阶段:（i） iPSC 在悬浮液中聚集形成分化为中胚层细胞的三维拟胚体 （EB），（ii） 从分化的 EB 形成巨噬细胞前体细胞，以及 （iii） 巨噬细胞前体细胞分化为成熟的功能性巨噬细胞?...

视频: 人诱导多能干细胞分化为巨噬细胞 - 概念

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

科研

JoVE Journal

发育生物学

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

患者外周血单核细胞中人心肌细胞的生成和扩增

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

人小胶质细胞样细胞:使用人突触体从诱导多能干细胞和 体外 活细胞吞噬测定中分化

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages

成績單

Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages