STEMCELL Technologies STEMdiff STEMdiff Branching Lung Organoid Kit
- 研究用
STEMdiff™ Branching Lung Organoid Kit（ST-100-0195）は、ヒト胚性幹（ES）および人工多能性幹（iPS）細胞から分岐肺オルガノイドを作製する無血清培地です。ヒト多能性幹細胞（hPSC）から4段階、すなわち 1）胚体内胚葉、2）前腸前部内胚葉、3）肺芽オルガノイド、4）分岐肺オルガノイドを経て、分枝した肺オルガノイドへと効率的かつ再現性よく分化します。
本品は、mTeSR™1（ST-85850）で維持したhPSCからの分化に最適化されています。得られるオルガノイドは、EPCAM、NKX2.1、SOX2、SOX9、MUC1、およびP63を発現する近位および遠位様の分枝気道上皮構造を発達させます。また、Matrigel®サンドイッチで28日を超えて維持すると、SFTPC、SFTPB、ABCA3などのより成熟した肺細胞マーカーの発現が上昇します。
本品に含まれる、長期培養（Matrigel®サンドイッチで28日以上）による分岐肺オルガノイド成熟に必要な構成品は、STEMdiff™ Branching Lung Organoid Maturation Kit（ST-100-0528）として単品でも購入いただけます。
2018/05/14 12:00の製品情報
本製品は研究目的にのみ使用し、人や動物の医療用・臨床診断用・食品用としては使用しないようにご注意ください。
製品の特長
STEMdiff™ Branching Lung Organoidをもちいて、hPSC由来の分枝した肺オルガノイドを効率的に作製できます
- 生体内の気道分枝形態形成と近位-遠位特異化を再現する、生理学的関連性の高いモデル系です
- ヒトES/iPS細胞株から分岐肺オルガノイドへと安定して分化できます
- 凍結保存可能な中間段階を備え、柔軟に実験できる便利なフォーマットです
- mTeSR™1との併用に最適化された、組成が明確な無血清培地です
分岐肺オルガノイド作製の流れ
(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.
データ紹介
分化段階での主要な肺マーカー発現
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).
分岐肺オルガノイドの主要マーカー発現と分枝形態
(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.
長期培養による成熟に伴う成熟肺マーカー発現上昇
(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.
オルガノイドの細胞が示す肺特異的な分化
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.
