Stemdiff smadi neural induction kit

Manufactured by STEMCELL

Sourced in Australia, Canada

The STEMdiff™ SMADi Neural Induction Kit is a cell culture product designed for the differentiation of pluripotent stem cells into neural progenitor cells. The kit contains the necessary components to efficiently generate neural progenitor cells from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs).

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Lab products found in correlation

Astrocyte medium, by ScienCell (6 mentions) Accutase, by STEMCELL (5 mentions) Stemdiff neural progenitor medium, by STEMCELL (4 mentions) Accutase, by Merck Group (3 mentions) Stemdiff neural induction medium smadi, by STEMCELL (2 mentions) Matrigel, by Corning (2 mentions) Countess 3, by Thermo Fisher Scientific (2 mentions) Stemdiff smadi neural induction media, by Thermo Fisher Scientific (2 mentions) Cryostor cs10, by STEMCELL (2 mentions) Fudeltagw rtta, by Addgene (2 mentions) Plv teto hngn2 egfp puro, by Addgene (2 mentions) Neural progenitor cell medium, by STEMCELL (1 mentions) Eb formation medium, by STEMCELL (1 mentions) Mtesr 1 complete medium, by STEMCELL (1 mentions) Picrotoxin, by Merck Group (1 mentions) Poly l ornithine, by Merck Group (1 mentions) Stemdiff neural rosette selection reagent, by STEMCELL (1 mentions) 37 μm reversible strainer, by STEMCELL (1 mentions) Mtesr1 media, by STEMCELL (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions)

32 protocols using stemdiff smadi neural induction kit

Radial Glia/Neural Stem Cell Generation

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Radial glia/neural stem cell was generated using STEMdiff™ SMADi Neural Induction Kit (Stemcell Technologies, 08581) following manufacturer’s instruction. Human iPSCs were dissociated into single cells and seeded on AggreWell 800 plates in EB formation medium (Stemcell Technologies, 05893) to initiate EB formation. After 24 h, EB formation medium was exchanged to neural induction medium with daily half medium change for 3–4 days. Next, EBs were collected and replated onto Matrigel-coated dishes and cultured in neural induction medium for another 5–7 days to induce neural rosette formation. Neural rosettes were isolated as a single-cell suspension and replated onto Matrigel-coated dishes in neural induction medium for another 2–3 days. Radial glia/neural stem cells were maintained and amplified in neural progenitor cell medium (Stemcell Technologies, 05833) for further experiments.

Zhao J., Ikezu T.C., Lu W., Macyczko J.R., Li Y., Lewis-Tuffin L.J., Martens Y.A., Ren Y., Zhu Y., Asmann Y.W., Ertekin-Taner N., Kanekiyo T, & Bu G. (2023). APOE deficiency impacts neural differentiation and cholesterol biosynthesis in human iPSC-derived cerebral organoids. Stem Cell Research & Therapy, 14, 214.

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Generation of HD and Healthy iPSC-derived NSCs

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Age and sex-matched iPSCs from an apparently healthy individual (GM23476, female, 20 years old at sampling, RRID:CVCL_T841) and a HD patient (GM23225, female, 20 years old at sampling, RRID:CVCL_F169, CAG repeat length 71) were obtained from MIGMS cell repository through Coriell Institute for Medical Research. iPSCs were cultured in Matrigel-coated plates with mTeSR^™1 complete medium (STEMCELL Technologies). The differentiation of iPSCs to NSCs was performed using the STEMdiff^™ SMADi Neural Induction Kit based on the rosette formation and isolation method according to the manufacture’s instruction (STEMCELL Technologies). After differentiation, NSCs were further cultured and expanded in the complete STEMdiff^™ Neural Progenitor Basal Medium (STEMCELL technologies). NSC expansions were limited to 4 passages. The use of human iPSC lines was approved by Institutional Biosafety Committee of Florida Atlantic University (Approval number B20-21). All iPSCs utilized in this study were used within 10 passages from cryopreserved stocks previously determined to be karyotypically normal. None of the cell lines used in this study (SH-SY5Y, STHdhQ7 and Q111, iPSCs) are listed as commonly misidentified cell lines by the International Cell Line Authentication Committee (ICLAC). No further authentication was performed in the laboratory.

Xu H., Bensalel J., Raju S., Capobianco E., Lu M.L, & Wei J. (2022). Characterization of huntingtin interactomes and their dynamic responses in living cells by proximity proteomics. Journal of neurochemistry, 164(4), 512-528.

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Differentiating hiPSCs into Cortical Neurons

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The hiPSC line LUMC0114iCTRL01 (hPSCreg number LUMCi003-A)^{44 (link)} was used to derive neural progenitor cells (NPCs) using the STEMdiff SMADi Neural Induction kit (05835, StemCell Technologies). The chip was plasma treated (50 W, 50 KHz, 45 s with CUTE Plasma System from Femto Science, Selangor, Malaysia) prior to coating with Poly-L-Ornithine (P3655,Sigma Aldrich) with a concentration of 100

μ

g/mL and incubated at room temperature (RT) overnight. The next day the chip was incubated at

4^{\circ} C

for 30 min before a laminin coating (

200 \frac{μ g}{mL}

) was applied. After which the chip was incubated at

37^{\circ} C

for 2 h. NPCs were seeded at a concentration of 100.000 cells

\cdot {cm}^{- 2}

on the chip and were subsequently differentiated into cortical neurons for 7 days using the STEMdiff Midbrain Neuron Differentiation Kit (100-0038, StemCell Technologies). Lastly, the hiPSC-derived cortical neurons were matured and maintained in BrainPhys hiPSC Neuron kit media (05795, StemCell Technologies) for the remainder of the experiment. All media were supplemented with

1 %

penicillin/streptovidin. For the drug experiments, to block inhibition, picrotoxin (P1675-1G, Sigma Aldrich) was prepared at a concentration of 50

μ M

in BrainPhys medium and was added 20 seconds after the recordings were started.

Aydogmus H., Hu M., Ivancevic L., Frimat J.P., van den Maagdenberg A.M., Sarro P.M, & Mastrangeli M. (2023). An organ-on-chip device with integrated charge sensors and recording microelectrodes. Scientific Reports, 13, 8062.

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Efficient Directed Differentiation of PSCs into NPCs

Cited 2 times

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Primed PSCs were differentiated into expandable NPCs by using the STEMdiff SMADi Neural Induction Kit (Stem Cell Technologies) as previously described [34 (link)–36 (link)]. In brief, primed PSCs were maintained on a Matrigel (Corning)-coated plate in mTeSR1 media (Stem Cell Technologies) prior to the NPC induction. The cells were harvested using Accutase (EMD Millipore) and transferred at 3 x 10⁶ cells to a well of an AgrreWell800 plate (Stem Cell Technologies) in STEMdiff Neural Induction Medium + SMADi (Stem Cell Technologies) supplemented with 10 μM Y-27632. Five days later, uniformly sized aggregates were collected using a 37 μm Reversible Strainer (Stem Cell Technologies) and plated onto a Matrigel-coated 6-well plate in STEMdiff Neural Induction Medium + SMADi. Seven days later, neural rosette structures were selectively removed by using STEMdiff Neural Rosette Selection Reagent (Stem Cell Technologies) and plated onto a new Matrigel-coated 6-well plate in STEMdiff Neural Induction Medium + SMADi. After that, the cells were passaged every 2–3 days until day 30 post-differentiation. The established NPCs were maintained on a Matrigel-coated plate in STEMdiff Neural Progenitor Medium (Stem Cell Technologies) and passaged every 3–4 days.

Takahashi K., Nakamura M., Okubo C., Kliesmete Z., Ohnuki M., Narita M., Watanabe A., Ueda M., Takashima Y., Hellmann I, & Yamanaka S. (2021). The pluripotent stem cell-specific transcript ESRG is dispensable for human pluripotency. PLoS Genetics, 17(5), e1009587.

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Generation of human neural progenitor cells

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Undifferentiated hiPSCs were cultured and maintained in mTeSR Plus medium (StemCell Technologies) on Matrigel (BD Biosciences)-coated plates prior to the generation of human neural progenitor cells (hNPCs). hNPCs were generated using the STEMdiff SMADi Neural Induction kit (StemCell Technologies) according to the manufacturer’s protocol. Briefly, neuralized embryoid bodies (EBs) were generated by culturing small aggregates of hESCs in ultra-low attachment plates (Corning) in STEMdiff SMADi Neural induction medium. EBs were replated and cultured in poly-D-lysine/Laminin-coated plates with the neural induction medium until EBs formed neural rosette formations. Neural rosettes were collected and replated in poly-D-lysine/Laminin-coated plates for hNPCs outgrowth in neural induction medium until hNPCs were ready for the first passage. hNPCs were maintained in STEMdiff Neural Progenitor medium. For neurogenesis of hNPCs, hNPCs were cultured in Neurobasal A medium supplemented with B27 minus vitamin A, 1% penicillin-streptomycin and glutamax (all from Invitrogen) in poly-D-lysine/Laminin-coated plates (Konopka et al., 2012 (link); Usui et al., 2017 (link)). For quantification, an average of 700 cells was counted per condition in 5–6 random fields per independent experiment.

Tomita H., Hines K.M., Herron J.M., Li A., Baggett D.W, & Xu L. (2022). 7-Dehydrocholesterol-derived oxysterols cause neurogenic defects in Smith-Lemli-Opitz syndrome. eLife, 11, e67141.

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Generation of Neural Progenitor Cells from Familial Alzheimer's Disease hiPSCs

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Patient-derived AD hiPSCs sourced from a male patient with an APP gene mutation causing fAD (ADAPP) and hiPSCs from an age-matched healthy female (HN1) were cultured in mTeSR^TM1 (STEMCELL Technologies cat no. 85850) on cell culture plates coated with Corning®Matrigel®. The hiPSCs were then passaged onto 6-well plates coated with 50 µg/mL of poly-L-ornithine (PLO, cat no. P4957, Sigma, St. Louis, MO, USA) and 10 µg/mL of laminin (cat no. L2020, Sigma, St. Louis, MO, USA). Neural induction was performed using a monolayer culture protocol and a STEMdiff™ SMADi Neural Induction Kit (STEMCELL Technologies cat no. 08581). Briefly, after the hiPSCs were plated as single cells onto 6-well plates, they were cultured for seven days at 37 °C and 5% CO₂ with daily media changes performed using STEMdiff™ Neural Induction Medium + SMADI. On day seven the cells were again passaged onto 6-well plates and daily media changes were performed with STEMdiff™ Neural Induction Medium + SMADI. This continued until passage three on day twenty-one when the cells were ready for expansion.

Benwood C., Walters-Shumka J., Scheck K, & Willerth S.M. (2023). 3D bioprinting patient-derived induced pluripotent stem cell models of Alzheimer’s disease using a smart bioink. Bioelectronic Medicine, 9, 10.

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Directed Differentiation of iPSCs into NPCs

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NPCs were differentiated using the STEMdiff™ SMADi Neural Induction Kit (STEMCELL Technologies) as monolayer cultures, according to the manufacturer’s recommendation. Two million iPSCs per well were plated on Matrigel^®-coated six-well plates (Corning) in a single-cell suspension using STEMdiff™ Neural Induction Medium + SMADi (STEMCELL Technologies), supplemented with 10 μM ROCK inhibitor (Stemgent). Daily medium changes were performed with STEMdiff™ Neural Induction Medium + SMADi (STEMCELL Technologies). Cells were passaged after 7 days using Accutase (STEMCELL Technologies) and plated at a density of 2 × 10⁶ cells per well. After 7 days of growth, cells were passaged for a total of two passages. Mature NPCs were then plated at a density of 1.2 × 10⁶ cells per well on Matrigel^®-coated six-well plates (Corning) and cultured in STEMdiff™ Neural Progenitor Medium (STEMCELL Technologies) with daily medium changes.

Schreiber A.M., Li Y., Chen Y.H., Napierala J.S, & Napierala M. (2022). Selected Histone Deacetylase Inhibitors Reverse the Frataxin Transcriptional Defect in a Novel Friedreich’s Ataxia Induced Pluripotent Stem Cell-Derived Neuronal Reporter System. Frontiers in Neuroscience, 16, 836476.

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Differentiation of Human iPSCs and ESCs into NPCs

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The human iPSC line was purchased from the Cell Inspire Biotechnology Co., Ltd. in Shenzhen, China, and maintained in mTeSR1™ completed medium (STEMCELL Technologies). The human ESC line H1 was purchased from the MeisenCTCC in Zhejiang, China, and maintained in mTeSR™ Plus completed medium (STEMCELL Technologies). The iPSC and ESC lines were cultured in 6-well plates coated with the Matrigel (Corning) diluted with 1% in DMEM/F12 (Gibco). Human NPC line ReNcell® VM (Sigma-Aldrich) was maintained in the ReNcell® NSC maintenance media (Sigma-Aldrich) supplied with 20 ng/mL basic fibroblast growth factor (bFGF) (CHEMICON) and 20 ng/mL epidermal growth factor (EGF) (CHEMICON). For NPC generation, we applied the STEMdiff™ SMADi Neural Induction Kit (STEMCELL Technologies) to induce the iPSCs to differentiate into NPCs following the manufactory protocol. The neural progenitors were maintained in the STEMdiff™ Neural Progenitor Medium (STEMCELL Technologies). The lentivirus solution (Hanbio) was incubated with the ReNcell to label the cells with EGFP. The cells that failed to transfect were eliminated by the treatment of puromycin (MCE). All the cells were maintained at 37°C in an incubator with a 5% CO₂ supply.

Wang Z., Zhao H., Tang X., Meng T., Khutsishvili D., Xu B, & Ma S. (2022). CNS Organoid Surpasses Cell-Laden Microgel Assembly to Promote Spinal Cord Injury Repair. Research, 2022, 9832128.

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Astrocyte Differentiation from iPSCs

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The human induced pluripotent stem cell (iPSC) line was purchased from Thermo Fisher (A18945). We followed our previous protocol used for the astrocyte differentiation from the SLC6A1(S295L) patient cell line (Mermer et al., 2021 (link)). We first prepared the neural progenitor cells (NPCs) and then differentiated the NPCs into astrocytes. The differentiation of NPCs was induced with the STEMdiff SMADi Neural induction kit from STEMCELL. Differentiation of human astrocytes started from NPCs at day 5 of passage 1 (P1). The differentiation of astrocytes was initiated by using the Astrocyte medium (ScienCell) for 25–30 days (doi: https://doi.org/10.1101/2020.04.21.054361) (Romero-Morales et al., 2021 (link)) and the culture medium was refreshed every other day. Astrocytes were passaged at ~70% confluence and were validated based on the protocol described in our previous study by staining with S100β and Glial Fibrillary Acidic Protein (GFAP) (Mermer et al., 2021 (link)). >95% of cells adopted astrocytic morphology and about 80% of the total cell population was positively stained with GFAP and S100B, consistent with previous studies on astrocyte differentiation (Mermer et al., 2021 (link)).

Mermer F., Poliquin S., Zhou S., Wang X., Ding Y., Yin F., Shen W., Wang J., Rigsby K., Xu D., Mack T., Nwosu G., Flamm C., Stein M, & Kang J.Q. (2022). Astrocytic GABA transporter 1 deficit in novel SLC6A1 variants mediated epilepsy: Connected from protein destabilization to seizures in mice and humans. Neurobiology of disease, 172, 105810.

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Efficient Neural Induction of iPSCs

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Differentiation was performed using the STEMdiff SMADi Neural Induction Kit (StemCell Technologies 08581) according to the manufacturer’s monolayer protocol. Briefly, at day 0 the four human iPSC clones (WT-A, WT-B, MT-A and MT-B) were plated at 2 × 10⁶ cells per well in a Geltrex-coated six-well plate with mTeSr1 medium. The medium was replaced with neural induction medium with SMADi the next day and daily medium changes were performed onwards. On day 8, cells were passaged at 1:6 radio to a new well with neural induction medium with SMADi. Immunofluorescence experiments were performed on day-8 and day-11 NPCs, using similar protocols as described in ‘CRISPR genome editing of iPSCs’ except with the following primary antibodies: anti-PAX6 (mouse, 1:200, ThermoFisher MA1109), anti-OCT4 (rabbit, 1:400, Cell Signaling Technologies 2840), anti-Nestin (mouse, 1:200, ThermoFisher MA1110) and anti-SOX1 (rabbit, 1:200, Novus Biologicals NBP2–24486SS).

Cooley L., Thompson D, & Spradling A.C. (1990). Constructing deletions with defined endpoints in Drosophila.. Proceedings of the National Academy of Sciences of the United States of America, 87(8), 3170-3173.

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