STEMdiff™ Branching Lung Organoid Kit

Serum-free media for generation and maturation of branching lung organoids from human ES and iPS cells
STEMdiff™ Branching Lung Organoid Maturation Kit

Serum-free media for maturation of branching lung organoids from human ES and iPS cells

1 Kit
Catalog # 100-0528
STEMdiff™ Branching Lung Organoid Kit

Serum-free media for generation and maturation of branching lung organoids from human ES and iPS cells

1 Kit
Catalog # 100-0195
Required Products
  1. mTeSR™1
    mTeSR™1

    cGMP, feeder-free maintenance medium for human ES and iPS cells

  2. Gentle Cell Dissociation Reagent
    Gentle Cell Dissociation Reagent

    cGMP, enzyme-free cell dissociation reagent

  3. DMEM/F-12 with 15 mM HEPES
    DMEM/F-12 with 15 mM HEPES

    Dulbecco's Modified Eagle's Medium/Nutrient Ham's Mixture F-12 (DMEM/F-12) with 15 mM HEPES buffer

  4. Costar® 24-Well Flat-Bottom Plate, Tissue Culture-Treated
    Costar® 24-Well Flat-Bottom Plate, Tissue Culture-Treated

    Clear polystyrene flat-bottom, tissue culture-treated multiwell cell culture plate with lid

Overview

STEMdiff™ Branching Lung Organoid Kit (Catalog #100-0195) is a serum-free medium system for efficient and reproducible generation of branching lung organoids from human embryonic stem (ES) and induced pluripotent stem (iPS) cells through four stages of differentiation: 1) definitive endoderm, 2) anterior foregut endoderm, 3) lung bud organoid, and 4) branching lung organoids. STEMdiff™ Branching Lung Organoid Kit has been optimized for differentiation of cells maintained in mTeSR™1 (Catalog #85850). Resulting organoids will develop proximal and distal-like branching airway epithelial structures expressing EPCAM, NKX2.1, SOX2, SOX9, MUC1, and P63. When maintained beyond 28 days in a Matrigel® sandwich, there is an increase in expression levels of markers for more mature lung cells, such as SFTPC, SFTPB, and ABCA3. For extended periods of organoid culture (> 28 days in the Matrigel® sandwich), the components required for branching lung organoid maturation are available in STEMdiff™ Branching Lung Organoid Maturation Kit (Catalog #100-0528).
Advantages
⦁ Physiologically relevant model system that recapitulates in vivo airway branching morphogenesis and proximo-distal specification
⦁ Robust medium supports efficient differentiation of human ES and iPS cell lines to branching lung organoids
⦁ Convenient format with cryopreservable intermediate stage for experimental flexibility
⦁ Defined and serum-free medium system optimized to work with mTeSR™1
Components
  • STEMdiff™ Endoderm Basal Medium (Lung), 100 mL
  • STEMdiff™ Definitive Endoderm Supplement MR (100X), 0.35 mL
  • STEMdiff™ Definitive Endoderm Supplement CJ (100X), 1.1 mL
  • STEMdiff™ Lung Basal Medium, 180 mL
  • STEMdiff™ Lung Supplement (10X), 20 mL
  • STEMdiff™ Branching Lung Organoid Supplement A (100X), 0.25 mL
  • STEMdiff™ Branching Lung Organoid Supplement B (100X), 1.75 mL
Subtype
Specialized Media
Cell Type
Epithelial Cells, PSC-Derived, Mesenchymal Cells, PSC-Derived, Pluripotent Stem Cells
Species
Human
Application
Differentiation, Genome Editing, Organoid Culture
Brand
STEMdiff
Area of Interest
Disease Modeling, Drug Discovery and Toxicity Testing, Infectious Diseases, Respiratory Research, Transplantation Research
Formulation
Serum-Free

Related Products

Scientific Resources

Product Documentation

Document Type Product Name Catalog # Lot # Language
Document Type
Product Information Sheet
Product Name
STEMdiff™ Branching Lung Organoid Maturation Kit
Catalog #
100-0528
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
STEMdiff™ Branching Lung Organoid Kit
Catalog #
100-0195
Lot #
All
Language
English
Document Type
Special Protocol
Product Name
STEMdiff™ Branching Lung Organoid Kit
Catalog #
100-0195
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
STEMdiff™ Branching Lung Organoid Maturation Kit
Catalog #
100-0528
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
STEMdiff™ Branching Lung Organoid Maturation Kit
Catalog #
100-0528
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
STEMdiff™ Branching Lung Organoid Maturation Kit
Catalog #
100-0528
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
STEMdiff™ Branching Lung Organoid Kit
Catalog #
100-0195
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
STEMdiff™ Branching Lung Organoid Kit
Catalog #
100-0195
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
STEMdiff™ Branching Lung Organoid Kit
Catalog #
100-0195
Lot #
All
Language
English
Document Type
Safety Data Sheet 4
Product Name
STEMdiff™ Branching Lung Organoid Kit
Catalog #
100-0195
Lot #
All
Language
English
Document Type
Safety Data Sheet 5
Product Name
STEMdiff™ Branching Lung Organoid Kit
Catalog #
100-0195
Lot #
All
Language
English
Document Type
Safety Data Sheet 6
Product Name
STEMdiff™ Branching Lung Organoid Kit
Catalog #
100-0195
Lot #
All
Language
English
Document Type
Safety Data Sheet 7
Product Name
STEMdiff™ Branching Lung Organoid Kit
Catalog #
100-0195
Lot #
All
Language
English

Educational Materials(12)

Brochure
Products for Human Pluripotent Stem Cells
Brochure
Organoids
Brochure
STEMdiff™ Definitive Endoderm Kit
Wallchart
Cellular Organization and Biology of the Respiratory System
Wallchart
Directed Differentiation of Pluripotent Stem Cells
Video
4:46
Supporting Science Globally: A look into how scientists in Singapore are using organoids
Webinar
1:10:09
HUB & STEMCELL Organoids as Models of Infectious Disease Mini-Symposium
Webinar
44:40
Making Lung Cells from Pluripotent Stem Cells: Disease Modeling and Future Therapies
Webinar
18:41
In Vitro Human Airway Modeling Using Primary Airway Epithelial Cells
Webinar
20:24
STEMdiff™ Kits for Robust and Efficient Differentiation of hPSCs to Multiple Cell Types
Webinar
59:52
ISSCR Innovation Showcase: Applications of Organoid and Organotypic Cultures in Infectious Diseases, Nephrotoxicity, and Highly Relevant Cell-Based Assay
Webinar
52:35
Organoid Expert Panel

Product Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Data and Publications

Data

Schematic showing the different stages in the STEMdiff™ Branching Lung Organoid Kit protocol and the representative morpholgy at each stage.

Figure 1. Generation Of Branching Lung Organoids Using the STEMdiff™ Branching Lung Organoid Kit

(A) Human PSC cultures progress through a four-stage differentiation process to generate human branching lung organoids. By the end of stage 1 (day 3), cultures exhibit characteristics typical of definitive endoderm and anterior foregut differentiation is initiated. During stage 2 (days 3 - 6) anterior foregut endoderm buds are released from the monolayer, and are then suspended to form ventralized lung bud organoids in stage 3 (days 6 - 14). In stage 4, the lung bud organoids are embedded into Matrigel sandwich cultures to mature into branching lung organoids. (B) Morphological representation of the culture at different stages.

Flow cytometry data at the end of stage 2 demonstrating expression of SOX2 and absence of CDX2 in AFE buds and brnaching lung organoids cultured beyond stage 4 demonstrating the expression of CPM and NKX2.1.

Figure 2. STEMdiff™ Branching Lung Organoid Kit Supports Expression of Key Lung Markers at Different Stages

Flow cytometric analysis at different stages of the STEMdiff™ Branching Lung Organoid Kit workflow shows protein expression of key lung markers. (A) Anterior foregut endoderm (AFE) buds generated at the end of stage 2 demonstrates high expression of the anterior foregut marker, SOX2 and absence of mid/hindgut marker, CDX2. (B) Cells from branching lung organoids on day 42 (stage 4) express lung progenitor marker NKX2.1 and its surrogate cell surface marker CPM. (C) NKX2.1 and CPM expression are maintained in long-term cultures as seen by flow cytometric analysis of cells from branching lung organoids (day 67).

Panel of immunocytochemistry (ICC) images demonstrating the protein expression of key lung markers in branching lung organoids generated using STEMdiff™ Branching Lung Organoid Kit and matured using STEMdiff™ Branching Lung Organoid Maturation Kit. Panel of immunocytochemistry (ICC) images demonstrating the protein expression of key lung markers in branching lung organoids generated using STEMdiff™ Branching Lung Organoid Kit and matured using STEMdiff™ Branching Lung Organoid Maturation Kit.

Figure 3. Branching Lung Organoids Cultured in STEMdiff™ Branching Lung Organoid Kit Feature Key Protein Markers and Exhibit Branching Morphogenesis

(A) Branching lung organoids express lung progenitor marker NKX2.1 throughout their branching structures, and (B, C) demonstrate the presence of alveolar type II-like cells with Pro-surfactant protein B and C expressions. (D, E) These organoids undergo proximodistal differentiation demonstrated by the differential expression of SOX2 and SOX9. (F) MUC1 can be found luminally expressed while the (G) organoids are surrounded by VIM-expressing mesenchyme. (H, I) Branching lung organoids generated in STEMdiff™ Branching Lung Organoid Kit also express proteins associated with SARS-CoV-2 entry, ACE2 and TMPRSS2. Protein expression was visualized by immunohistochemistry and confocal microscopy of branching lung organoids on day 63.

Figure 4. Branching Lung Organoids Cultured in STEMdiff™ Branching Lung Organoid Maturation Kit For Extended Periods Express More Mature Lung Markers

(A) The branching tips of branching lung organoids derived from peripheral blood endothelial progenitor cell-derived iPS cells (iPSC-1), peripheral blood mononuclear cell-derived iPS cells (iPSC-2) or embryonic stem cells (H1 and H9) continue to grow and branch up to day 101. (B) The expression levels of more mature distal lung markers ABCA3, SFTPC, and SFTPB increase overtime. Morphology and gene expression of branching lung organoids cultured up to day 105 were assessed by RT-qPCR. RQ-values were normalized to TBP and compared to commercially available lung RNA.

Gene expression data of branching lung organoids of key lung markers like NKX2.1, MUC1, P63, FOXJ1, SFTB, SFTC, and ABCA3 and the absence of off-target markers like PAX8 and FOXG1.

Figure 5. Branching Lung Organoid Cells Are Specifically Differentiated Towards the Lung

Gene expression profile of branching lung organoids (day 42+) analysed by RT-qPCR show presence of key lung markers and absence of off-target markers. Branching lung organoids at day 42 express (A) lung-thyroid-forebrain marker NKX2.1 and does not exhibit thyroid marker PAX8 and forebrain marker FOXG1. (B) The expressions of proximal lung markers MUC1, P63, and FOXJ1 and distal lung markers SFTPC, SFTPB, and ABCA3 are upregulated in day 56 branching lung organoids, when compared to respective pluripotent stem cells (PSCs). All expression levels are normalized to the housekeeping gene TBP. iPSC-1: peripheral blood endothelial progenitor cell-derived iPS cells; iPSC-2: peripheral blood mononuclear cell-derived iPS cells.

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