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Stemdiff cardiomyocyte differentiation kit

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The STEMdiff Cardiomyocyte Differentiation Kit is a laboratory product designed to facilitate the differentiation of human pluripotent stem cells into cardiomyocytes. The kit provides a defined, serum-free culture system that supports the stepwise differentiation of stem cells into functional cardiomyocytes.

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Lab products found in correlation

Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions) Cardiomyocyte dissociation media, by STEMCELL (3 mentions) Mtesr1 medium, by STEMCELL (3 mentions) Hesc qualified matrigel, by Corning (3 mentions) Stemdiff cardiomyocyte maintenance medium, by STEMCELL (2 mentions) Y 27632, by STEMCELL (2 mentions) Matrigel, by Corning (2 mentions) Human pluripotent stem cell functional identification kit, by R&D Systems (2 mentions) Gm09503, by Coriell Institute for Medical Research (2 mentions) Gm25313, by Coriell Institute for Medical Research (2 mentions) Gm04569, by Coriell Institute for Medical Research (2 mentions) Gm05089, by Coriell Institute for Medical Research (2 mentions) Gm02298, by Coriell Institute for Medical Research (2 mentions) Gm04981, by Coriell Institute for Medical Research (2 mentions) Dl lactate solution, by Merck Group (1 mentions) Lipofectamine ltx transfection reagent, by Thermo Fisher Scientific (1 mentions) Pmd2 g vsvg, by Addgene (1 mentions) Pspax2, by Addgene (1 mentions) 5α dihydrotestosterone, by Merck Group (1 mentions) Ifn γ, by Thermo Fisher Scientific (1 mentions)

17 protocols using stemdiff cardiomyocyte differentiation kit

1

Cardiotoxicity Gene Role Assessment

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To ascertain the functional role of the prioritized genes in cardiotoxicity, human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) were generated. Two previously published hiPSC cell lines (PGP17_11 and PGP14_26), reprogrammed from lymphocytes taken from 2 healthy male donors in the Personal Genome Project (PGP) were used (25 (link)). We confirmed the absence of pathogenic or likely pathogenic SNVs (based on American College of Medical Genetics criteria) in known cardiotoxicity genes and genes identified in our study in the donors through interrogation of vcf files downloaded from the PGP-Canada website (26 (link)). The STEMdiff Cardiomyocyte Differentiation Kit (STEMCELL Technologies, Vancouver, Canada) was used to differentiate hiPSCs into CMs in accordance with our previously published differentiation protocol (26 (link)). At day 16, cells were dissociated, reseeded, and maintained in STEMdiff Cardiomyocyte Maintenance medium (STEMCELL Technologies), which was replaced every 2 days, in 80% relative humidity levels and 5% CO2 levels, to generate beating CMs.
Chaix M.A., Parmar N., Kinnear C., Lafreniere-Roula M., Akinrinade O., Yao R., Miron A., Lam E., Meng G., Christie A., Manickaraj A.K., Marjerrison S., Dillenburg R., Bassal M., Lougheed J., Zelcer S., Rosenberg H., Hodgson D., Sender L., Kantor P., Manlhiot C., Ellis J., Mertens L., Nathan P.C, & Mital S. (2020). Machine Learning Identifies Clinical and Genetic Factors Associated With Anthracycline Cardiotoxicity in Pediatric Cancer Survivors. JACC: CardioOncology, 2(5), 690-706.
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2

Differentiation of hiPSCs into Cardiomyocytes

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FA-HCM-affected hIPSCs and controls were differentiated into cardiomyocytes (hIPSC-CMs) using STEMdiff™ Cardiomyocyte Differentiation Kit (# 05010, STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s instructions. hIPSCs were seeded on Matrigel coated plates with mTESR plus medium and cultured until they were ~ 95% confluent. Then, the medium was changed to STEMdiff™ Cardiomyocyte Differentiation Medium A containing matrigel (1:100) for two days, followed by STEMdiff™ Cardiomyocyte Differentiation Medium B for two days, and STEMdiff™ Cardiomyocyte Differentiation Medium C for four days. hIPSC-CM were maintained in STEMdiff™ Cardiomyocyte Maintenance Medium, which was changed every other day. After eight days of differentiation, beating cells with embryonic cardiomyocyte morphology and expression of cardiomyocyte markers were observed in the wells (data not shown). hIPSC-CM were maintained in STEMdiff™ Cardiomyocyte Maintenance Medium for 30 days before further experiments were performed. This media was changed every other day. For downstream experiments, hIPSC-CM were dissociated with cardiomyocyte dissociation media (# 05025, STEMCELL Technologies, Vancouver, Canada).
Chidipi B., Angulo M.B., Shah S.I., Rieser M., Ullah G., McDonald T.V, & Noujaim S.F. (2021). The Dynamin Related Protein 1 is Decreased and the Mitochondrial Network is Altered in Friedreich’s Ataxia Cardiomyopathy. The international journal of biochemistry & cell biology, 143, 106137.
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3

Cardiomyocyte Differentiation and Analysis

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Cardiomyocytes were differentiated using STEMdiff Cardiomyocyte Differentiation kit (Stem Cell Technologies) as per the manufacturer’s instructions. This kit produces cardiomyocytes within 15 days that can be observed beating from around day 8. Cells were cultured in three ways: in conventional 24-well plates for Western blot and immunofluorescence, 24-well microelectrode array (MEA) plates (Axion BioSystems) for electrophysiological analysis, and 8-well SeaHorse XFp plates for mitochondrial function analysis. One day before differentiation, single cell suspensions of the iPSCs were plated to be > 90% confluent on the following day in mTeSR1 media supplemented with 10 μM Y-27632 (Stem Cell Technologies). Cardiomyocyte differentiation was carried out over eight days with media changes every two days using supplement A for days 1–2, supplement B for days 3–4 and supplement C for days 5–8. Eight days after inducing differentiation, cardiomyocytes were transferred into STEMdiff Cardiomyocyte Maintenance Medium (Stem Cell Technologies) and given half-media changes daily until 14 days old (21 days old for the electrophysiology studies).
Wood A.R., Foliaki S.T., Groveman B.R., Walters R.O., Williams K., Yuan J., Zou W.Q, & Haigh C.L. (2022). Hereditary E200K mutation within the prion protein gene alters human iPSC derived cardiomyocyte function. Scientific Reports, 12, 15788.
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4

iPS Cell to Cardiomyocyte Differentiation

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All iPS cell lines were transitioned to and maintained on growth factor-reduced basement membrane matrix Matrigel (Corning #356231) in mTeSR-1 medium. For directed cardiomyocyte differentiations, iPS cells were dissociated using Accutase and seeded onto 6WP or 12WP. The culture was allowed to reach 80–90% confluency and induced with the Stemdiff Cardiomyocyte Differentiation Kit (Stemcell Technologies #05010), according to the manufacturer’s instructions. Starting on day 7, differentiations were monitored daily for beating cardiomyocytes and onset of beating was recorded as the day when beating was first observed.
Kathiriya I.S., Rao K.S., Iacono G., Devine W.P., Blair A.P., Hota S.K., Lai M.H., Garay B.I., Thomas R., Gong H.Z., Wasson L.K., Goyal P., Sukonnik T., Hu K.M., Akgun G.A., Bernard L.D., Akerberg B.N., Gu F., Li K., Speir M.L., Haeussler M., Pu W.T., Stuart J.M., Seidman C.E., Seidman J.G., Heyn H, & Bruneau B.G. (2020). Modeling human TBX5 haploinsufficiency predicts regulatory networks for congenital heart disease. Developmental cell, 56(3), 292-309.e9.
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5

Cardiomyocyte Differentiation and Purification

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Patient-derived and control iPSC lines were differentiated into iCMs using STEMdiff™ Cardiomyocyte Differentiation Kit (STEMCELL Technologies), as per the manufacturer’s protocol. Briefly, iPSCs were seeded on matrigel-coated 12-well plates and reached >95% confluency before starting differentiation. Cells were then treated with STEMdiff™ Cardiomyocyte Differentiation Medium A containing matrigel (1:100) for 2 days, STEMdiff™ Cardiomyocyte Differentiation Medium B for 2 days, STEMdiff™ Cardiomyocyte Differentiation Medium C for 4 days, and STEMdiff™ Cardiomyocyte Maintenance Medium. After 15 days of differentiation, cardiomyocytes were maintained in RPMI 1640 medium (Thermo Fisher) supplemented with B27 (Thermo Fisher) until day 30. Purification of cardiomyocytes was achieved by a metabolic-selection method as previously described (Sharma et al., 2015 (link)) with glucose starvation (RPMI-glucose + B27) for 5 days before functionality analysis. Fresh media was renewed every other day. Cardiomyocyte aggregates were dissociated using cardiomyocyte dissociation media (STEMCELL Technologies) for downstream experiments.
Yang J., Argenziano M.A., Burgos Angulo M., Bertalovitz A., Beidokhti M.N, & McDonald T.V. (2021). Phenotypic Variability in iPSC-Induced Cardiomyocytes and Cardiac Fibroblasts Carrying Diverse LMNA Mutations. Frontiers in Physiology, 12, 778982.
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6

Generation of hiPSC-Derived Muscle Cells

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De-identified fresh frozen human skeletal muscle tissues were purchased from ProteoGenex Inc. The study protocol was approved by the Institutional Review Board of MD Anderson Cancer Center, University of Texas. Clinical information is summarized in Supplementary Table 1.
All hiPSC studies were approved by HEIP Stem Cell committee of the University of Texas, MD Anderson Cancer Center. De-identified human BMD patient’s donor fibroblasts cells (GM04569, GM05089, GM02298 and GM04981) and healthy donor fibroblast cells (GM09503) were obtained from Coriell Institute and reprogramed to iPS cells at Human Stem Cell Core (Baylor College of Medicine). The human DMD patients donor iPS cells (GM25313) were obtained from Coriell Institute. Clinical information is summarized in Supplementary Table 1. IPS cells were cultured on hESC-Qualified Matrigel (Corning) coated plates and maintained in feeder-free mTeSR™1 medium (Stemcell Technologies). The pluripotency of these iPSCs were verified using Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, Catalog # SC027). Antibodies with dilution information are listed in Supplementary Table 10. hiPS cells were differentiated to cardiomyocytes and skeletal muscle cells using the STEMdiff™ Cardiomyocyte Differentiation Kit (Stemcell Technologies) and skeletal muscle differentiation protocol previously described54 (link).
Zhang Y., Li Y., Hu Q., Xi Y., Xing Z., Zhang Z., Huang L., Wu J., Liang K., Nguyen T.K., Egranov S.D., Sun C., Zhao Z., Hawke D.H., Li J., Sun D., Kim J.J., Zhang P., Cheng J., Farida A., Hung M.C., Han L., Darabi R., Lin C, & Yang L. (2020). LncRNA H19 Alleviates Muscular Dystrophy Through Stabilizing Dystrophin. Nature cell biology, 22(11), 1332-1345.
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7

Hypoxia-Induced Cardiac Differentiation

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We purchased the human-induced pluripotent stem cell (iPSC) line del-AR1034ZIMA 001 from Allele Biotechnology (San Diego, California). The cells were differentiated under normal O2 conditions and maintained with the STEMdiff Cardiomyocyte Differentiation Kit (STEMCELL Technologies, Vancouver, British Columbia, Canada) according to the manufacturer’s instructions. After 15-day induction, approximately 90% of the cells were beating and positive for both cardiac troponin T and sarcomeric α-actinin. We re-seeded the cells and cultured them in different O2 concentrations (21%, 15%, 10%, and 5%) in incubators for 7 days. To produce rapid changes in O2, we incubated cells at 21% O2 for 2 days, and then changed the O2 level to 15% for 2 days, 10% for 2 days, and finally, 5% for 2 days. After 7 days of culture, we subjected the cells to DNA extraction, qPCR, and IF.
Ye L., Qiu L., Feng B., Jiang C., Huang Y., Zhang H., Zhang H., Hong H, & Liu J. (2020). Role of Blood Oxygen Saturation During Post-Natal Human Cardiomyocyte Cell Cycle Activities. JACC: Basic to Translational Science, 5(5), 447-460.
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8

Generating Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

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Human induced pluripotent stem cells (h-iPSCs) were generated and cultured as previously described [49 (link)]. In brief, h-iPSCs were generated through non-integrating episomal overexpression of reprogramming factors OCT4, SOX2, KLF4, L-MYC, and LIN28 into human MSCs via electroporation. H-iPSCs were cultured on Matrigel (Corning, Cat No. 354277) coated plate in mTeSR1 medium (STEMCELL, Cat No. 85850) and passaged when reaching 80% confluency. Cardiac differentiation was induced using STEMdiff™ cardiomyocyte differentiation kit (STEMCELL, Cat No. 05010) according to the manufacturer’s protocol. Following 8 days of differentiation, metabolic selection was performed with lactate medium composed of RPMI 1640 medium (no glucose, ThermoFisher, Cat No. 11879020) supplemented with 1% sodium DL-lactate solution (60%, Sigma, Cat No. L4263) and B27 (minus insulin, ThermoFisher, Cat No. A1895601). After 2 days of lactate selection, purified iCMs were maintained in STEMdiff™ cardiomyocyte maintenance medium.
Hong X., Oh N., Wang K., Neumeyer J., Lee C.N., Lin R.Z., Piekarski B., Emani S., Greene A.K., Friehs I., del Nido P.J, & Melero-Martin J.M. (2021). Human endothelial colony-forming cells provide trophic support for pluripotent stem cell-derived cardiomyocytes via distinctively high expression of neuregulin-1. Angiogenesis, 24(2), 327-344.
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9

Cardiac Differentiation of Stem Cells

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The process of cardiac differentiation was conducted using the STEMdiff Cardiomyocyte Differentiation Kit (STEM CELL Technologies), following the instructions outlined in the manual. Initially, hESCs or hiPSCs were dissociated and then re-seeded onto Matrigel-coated 12-well plates. Once the cells reached full confluence, they were exposed to differentiation basal medium supplemented with supplement A. After 48 h, the medium was substituted with differentiation basal medium containing supplement B. Moving forward, on day 4, the cells were incubated in differentiation basal medium along with supplement C. This medium was refreshed every 2 days. Upon reaching day 8, the cells were maintained in STEMdiff™ cardiomyocyte maintenance medium supplemented with supplement M. Similar to previous stages, this medium was renewed every 2 days. For experiments focused on human cardiac development, cells at specific time points in the differentiation process were collected as dictated by the experimental designs.
Liu J., Chen S., Huang G., Wen P., Zhou X, & Wu Y. (2024). Trisomy 21-driven metabolite alterations are linked to cellular injuries in Down syndrome. Cellular and Molecular Life Sciences: CMLS, 81(1), 112.
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10

Lentiviral Transduction of hiPSC-Derived Cardiomyocytes

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Lentiviruses were produced by transfection of HEK293T cells with pLVX-EIF1α-HA-GST- RagD-WT, pLVX-EIF1α-HA-GST-RagD-P88L and pLVX-EIF1α-HA-GST-RagD-S76L, constructs in combination with the psPAX2 [Addgene, #12260], pMD2G/VSVG [Addgene, #12259] packaging plasmids using Lipofectamine LTX transfection reagent (Invitrogen). Five hours after transfection, the medium was changed to DMEM supplemented with 10% FBS. Forty-eight hours later, virus-containing supernatants were collected, passed through a 0.45-μm filter to eliminate cell debris. hiPSCs (CBiPS1sv-4F-40, derived from cord blood of a neonatal female individual, registered at https://hpscreg.eu/cell-line/ESi007-A and supplied by EBiSC-European bank for induced pluripotent stem cells https://ebisc.org/ESi007-A) were cultured on human recombinant truncated vitronectin protein and cultured in E8 medium; cells were passaged twice a week with PBS containing EDTA 0.5 mM.
Cells were then differentiated into cardiomyocytes using the STEMdiff Cardiomyocyte Differentiation Kit (Stem Cell Technologies, #05010) and subjected to lentiviral infection (Multiplicity Of Infection: 3) at Day 13 (beating appeared at day 8) of differentiation in the presence of 8 μg/ml polybrene (cat. no. tr-1003-G, EMD Millipore).
Sambri I., Ferniani M., Campostrini G., Testa M., Meraviglia V., de Araujo M.E., Dokládal L., Vilardo C., Monfregola J., Zampelli N., Vecchio Blanco F.D., Torella A., Ruosi C., Fecarotta S., Parenti G., Staiano L., Bellin M., Huber L.A., De Virgilio C., Trepiccione F., Nigro V, & Ballabio A. (2023). RagD auto-activating mutations impair MiT/TFE activity in kidney tubulopathy and cardiomyopathy syndrome. Nature Communications, 14, 2775.
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