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Stemdiff neural system

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The STEMdiff neural system is a laboratory equipment product that supports the differentiation of pluripotent stem cells into neural lineages. It provides a defined, serum-free culture system for the efficient generation of neural progenitor cells and mature neural cell types. The core function of this system is to facilitate the controlled, stepwise differentiation of stem cells towards specific neural cell fates.

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Lab products found in correlation

Aggrewell 800 plate, by STEMCELL (2 mentions) Stemdiff neural rosette selection reagent, by STEMCELL (1 mentions) Stemdiff neural progenitor freezing medium, by STEMCELL (1 mentions)

7 protocols using stemdiff neural system

1

Directed Neural Differentiation of iPSCs

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Human NPC differentiation from monolayer iPSCs was accomplished using the STEMdiff neural system according to the manufacturer's instructions (Stem Cell Technologies). Briefly, on day 0, human iPSCs maintained in mTESR1 medium on Matrigel-coated plates were dissociated and plated in STEMdiff neural induction medium + 10 µM Y-27632. The medium was changed, and cells were split (as required per the manufacturer's instructions) until day 6. On day 6, the medium was changed to STEMdiff neural progenitor medium until the completion of differentiation. Time points were collected every 3 d for RNA and immunofluorescence staining.
Ferrer C.M., Alders M., Postma A.V., Park S., Klein M.A., Cetinbas M., Pajkrt E., Glas A., van Koningsbruggen S., Christoffels V.M., Mannens M.M., Knegt L., Etchegaray J.P., Sadreyev R.I., Denu J.M., Mostoslavsky G., van Maarle M.C, & Mostoslavsky R. (2018). An inactivating mutation in the histone deacetylase SIRT6 causes human perinatal lethality. Genes & Development, 32(5-6), 373-388.
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2

Generation and Maintenance of Human iPSC-Derived Neural Progenitor Cells

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Cellartis® Human iPSC Lines from Takara (ChiPSC22, Cat. No. Y00320) were cultured with strict adherence to manufacturer’s protocols and manuals. Cellartis® DEF-CS 500 (Y30017) culture system was employed to maintain iPSC cultures (thawing, passages, media changes and cryopreservation).
Generation and culturing of Neural Progenitor Cells (NPCs) was achieved with the STEMdiff® Neural System from STEMCELL Technologies. Briefly, STEMdiff® SMADi Neural Induction kit (Cat. No. 08581) was used to treat iPSC in culture according to manufacturer’s protocol that generates CNS-type NPCs. Following induction, NPC cultures were maintained with STEMdiff® Neural Progenitor Medium system (Cat. No. 05833). We performed the induction and selection according to the “monolayer culture protocol”. We considered Day 10 post-NPC induction as early-stage NPCs and day 29 as late-stage NPCs. Pellets were collected and population doublings were determined post-differentiation (~Day 15–20).
Kim J.J., Sayed M.E., Ahn A., Slusher A.L., Ying J.Y, & Ludlow A.T. (2023). Dynamics of TERT regulation via alternative splicing in stem cells and cancer cells. PLOS ONE, 18(8), e0289327.
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3

Differentiation of hESCs into NPCs

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Human ESCs were differentiated into neural progenitor cells (NPCs) using the STEMdiff neural system (STEMCELL Technologies). Briefly, 2-3 X 106 hESCs suspended in the neural induction medium with the ROCK inhibitor Y27632 were seeded on AggreWell 800 plates (STEMCELL Technologies) for embryoid body (EB) formation. The neural induction medium was changed every day for 5 days. EBs were harvested on day 5 using 37-μm reversible strainers. Collected EBs were replated on fresh Matrigel-coated plates and incubated for 7 days to allow neural rosette (NR) formation. NRs were then selected using STEMdiff NR selection reagent. Selected cells were replated and allowed to grow for another 4-6 days in neural induction medium to form neural progenitor cells (NPCs). NPCs were passaged in STEMdiff neural progenitor medium.
Sivakumar S., Qi S., Cheng N., Sathe A.A., Kanchwala M., Kumar A., Evers B.M., Xing C, & Yu H. (2022). TP53 promotes lineage commitment of human embryonic stem cells through ciliogenesis and sonic hedgehog signaling. Cell reports, 38(7), 110395.
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4

Neural Differentiation of Human Embryonic Stem Cells

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Human embryonic stem cells (H1 and H9 lines) obtained through license agreement with WiCell Research Institute (Madison, WI) were cultured on mouse embryonic fibroblast (MEF) feeder layer and were transferred to mTeSR1 serum free human embryonic stem cell (hESC) culture system (STEMCELL Technologies Inc., Vancouver, Canada). Neural differentiation of hESCs was performed by using STEMdiff Neural System (STEMCELL Technologies Inc., Vancouver, Canada) according to the manufacturer’s instruction as described in our previous publications [23 (link), 24 (link)]. After 7 day differentiation, morphological assessment and scoring of neural rosettes were done to ensure 50% or more of the area of each aggregate was filled with neural rosettes (as shown in Fig 1). On day 7, neural rosettes were selected away from contaminating flat cells and collected. The rosettes were resuspended in pre-warmed NIM and cultured on 6-well plates precoated with poly-L-Ornithine and laminin (PLO/L) with daily full medium changes using pre-warmed STEMdiff NIM (without or with 20 mM ethanol) for 5 days with alternating treatment for a day and withdrawal for a day. Cells were EtOH concentration was chosen for its physiological relevance in that 20 mM is equivalent to DUI level and 50 mM falls within levels measured in alcoholics [25 (link)].
Kim Y.Y., Roubal I., Lee Y.S., Kim J.S., Hoang M., Mathiyakom N, & Kim Y. (2016). Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells. PLoS ONE, 11(9), e0163812.
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5

hESC Differentiation into Neural Progenitors

Cited 1 time
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hESCs were differentiated into NPCs using adherent culture conditions (Chambers et al., 2009 (link)). We followed the monolayer culture protocol from STEMdiff™ Neural System (Stem cell technologies). Briefly, hESC monolayers were plated onto matrigel coated plates, and cells were incubated with neural induction medium for 13 days, followed by incubation with neural progenitor medium to maintain and expand NPCs (Figure S7A). Gene expression of MDA5, RIG-I, and PKR was knocked-down in NPCs as shown in Figure S7F. Briefly, a monolayer of hESCs were plated onto matrigel coated plates and incubated with neural induction medium. After 8 days, cells were split (50% confluency) into matrigel coated plates. The next day, cells were transduced with lentiviruses produced from pGIPZ shRNA lentiviral vectors (Dharmacon) – targeting MDA5, RIG-I, and PKR - and were further incubated with neural induction medium for 5 days before harvest.
Chung H., Calis J.J., Wu X., Sun T., Yu Y., Sarbanes S.L., Dao Thi V.L., Shilvock A.R., Hoffmann H.H., Rosenberg B.R, & Rice C.M. (2018). Human ADAR1 prevents endogenous RNA from triggering translational shutdown. Cell, 172(4), 811-824.e14.
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6

Differentiation of hESCs into NPCs

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Human ESCs were differentiated into neural progenitor cells (NPCs) using the STEMdiff neural system (STEMCELL Technologies). Briefly, 2-3 X 106 hESCs suspended in the neural induction medium with the ROCK inhibitor Y27632 were seeded on AggreWell 800 plates (STEMCELL Technologies) for embryoid body (EB) formation. The neural induction medium was changed every day for 5 days. EBs were harvested on day 5 using 37-μm reversible strainers. Collected EBs were replated on fresh Matrigel-coated plates and incubated for 7 days to allow neural rosette (NR) formation. NRs were then selected using STEMdiff NR selection reagent. Selected cells were replated and allowed to grow for another 4-6 days in neural induction medium to form neural progenitor cells (NPCs). NPCs were passaged in STEMdiff neural progenitor medium.
Sivakumar S., Qi S., Cheng N., Sathe A.A., Kanchwala M., Kumar A., Evers B.M., Xing C, & Yu H. (2022). TP53 promotes lineage commitment of human embryonic stem cells through ciliogenesis and sonic hedgehog signaling. Cell reports, 38(7), 110395.
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7

Generating Neural Progenitor Cells from iPSCs

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EBs that represent three embryonic germ layers were generated from the iPSC lines passage no. 14–18. The neural induction medium (05893, STEMCELL Technologies) and AggreWell™ 800 microwell culture plates (34815, STEMCELL Technologies) were used to form EBs by following step by step the commercially available protocol “STEMdiff™ neural system to generation and culture of neural progenitor cells” of STEMCELL Technologies. Practically, AggreWell™ plates allow for controlling the EBs’ size and having a uniform population. Once EBs were generated, they were transferred into a six-well plate to form rosettes. After 4–5 days, the rosettes were isolated using STEMdiff™ neural rosette selection reagent (05832, STEMCELL Technologies) and transferred into six-well plates to generate neuronal progenitor cells (NPCs). Once NPCs got around 80%–90% confluence, they were passaged and collected using accutase (07922, STEMCELL Technologies) and cryopreserved with STEMdiff™ neural progenitor freezing medium (05838, STEMCELL Technologies) in liquid nitrogen for further uses.
Patel N., Ouellet V., Paquet-Mercier F., Chetoui N., Bélanger E., Paquet M.E., Godin A.G, & Marquet P. (2023). A robust and reliable methodology to perform GECI-based multi-time point neuronal calcium imaging within mixed cultures of human iPSC-derived cortical neurons. Frontiers in Neuroscience, 17, 1247397.
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