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Relesr dissociation reagent

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ReLeSR is a dissociation reagent used for the gentle dissociation of human pluripotent stem cells (hPSCs) into a single-cell suspension. It is designed to maintain high cell viability and preserve the undifferentiated state of the cells.

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Lab products found in correlation

Stemdiff hematopoietic kit, by STEMCELL (4 mentions) Antibiotic antimycotic, by Thermo Fisher Scientific (3 mentions) Matrigel, by BD (3 mentions) Karyostat assay, by Thermo Fisher Scientific (2 mentions) Complete mtesr plus, by STEMCELL (2 mentions) Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions) Bambanker media, by Fujifilm (1 mentions) Stemflex medium, by Thermo Fisher Scientific (1 mentions) Jetprime reagent, by Polyplus Transfection (1 mentions)

7 protocols using relesr dissociation reagent

1

Hematopoietic Differentiation of iPSCs

Cited 2 times
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iPSCs were obtained from the iPSC repository of the DIAN project, directed by Dr. Karch (Washington University, St. Louis, MO, USA) [49 (link)]. Donor line information is detailed in Table 1. iPSC lines were thawed into Matrigel-coated 6-well plates and maintained by feeding every other day with Stemflex supplemented media (Thermo Fisher Scientific, Waltham, MA, US) at 37 °C, 5% CO2. When 70–80% confluence was reached, iPSCs were passaged using ReLeSR dissociation reagent (#05872, STEMCELL Technologies, Vancouver, Canada). Hematopoietic stem cells were differentiated from iPSCs using the STEMdiff Hematopoietic kit (#05310 STEMCELL Technologies, Vancouver, Canada,) according to a previously published protocol [41 (link)]. When cultured for a minimum of 10 days, HPCs were collected and, if viability was >75%, frozen in Bambanker media (Wako Chemicals, Osakan, Japan).
Romero-Molina C., Neuner S.M., Ryszawiec M., Pébay A., Marcora E, & Goate A. (2024). Autosomal Dominant Alzheimer’s Disease Mutations in Human Microglia Are Not Sufficient to Trigger Amyloid Pathology in WT Mice but Might Affect Pathology in 5XFAD Mice. International Journal of Molecular Sciences, 25(5), 2565.
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2

Maintaining human iPSCs in mTESR media

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CiPS001–13, male hiPS cells generously donated by Drs. Kevin Bersell and Dan Roden (Vanderbilt University) were maintained in mTESR 1 media (StemTechnologies 8580) supplemented with 1% Antibiotic-Antimycotic (Life Technologies 15240062) on BD hESC- qualified Matrigel (Fisher 08–774-552 Corning 354277 diluted according to the manufacturer’s COA). Cells were passaged every 5 days using ReLeSR dissociation reagent (Stem Cell Technologies 5872) according to the manufacturer’s protocol. 10 μM of Rho-associated kinase inhibitor (ROCK) Y27632 (EMD 68000) was added to the media for the first 24 hours after passaging. iPS cells grown at 37oC 5% CO2 and were maintained in 2 mL of mTESR media with changes every 48 hours until day of passage. The cell line has not been authenticated.
Han L., Choudhury S., Mich-Basso J.D., Ammanamanchi N., Ganapathy B., Suresh S., Khaladkar M., Singh J., Maehr R., Zuppo D.A., Kim J., Eberwine J.H., Wyman S.K., Wu Y, & Kühn B. (2020). Lamin B2 levels regulate polyploidization of cardiomyocyte nuclei and myocardial regeneration. Developmental cell, 53(1), 42-59.e11.
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3

Maintenance of Human iPSCs in mTESR

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CiPS001-13 iPSCs generously donated by Drs. Kevin Bersell and Dan Roden (Vanderbilt University) were maintained in mTESR one medium (StemTechnologies 85850) supplemented with 1% Antibiotic-Antimycotic (Life Technologies 15240062) on BD hESC-qualified Matrigel (Corning 354277). Cells were passaged every 5–6 days using ReLeSR dissociation reagent (Stem Cell Technologies 5872) according to the manufacturer’s protocol. Then, 10 mM of Rho-associated kinase inhibitor (ROCK) Y27632 (EMD 68000) was added to the medium for the first 24 h after passaging. Human iPSCs were incubated at 37°C in 5% CO2 and maintained in 2 ml of mTESR one medium with changes every 48 h until the day of passage.
Wang J., Morgan W., Saini A., Liu T., Lough J, & Han L. (2022). Single-cell transcriptomic profiling reveals specific maturation signatures in human cardiomyocytes derived from LMNB2-inactivated induced pluripotent stem cells. Frontiers in Cell and Developmental Biology, 10, 895162.
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4

Maintenance of iPS Cell Line

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CiPS001–13 iPS cells generously donated by Drs. Kevin Bersell and Dan Roden (Vanderbilt University) were maintained in mTESR 1 media (StemTechnologies 85850) supplemented with 1% Antibiotic-Antimycotic (Life Technologies 15240062) on BD hESC-qualified Matrigel (Fisher 08–774-552 Corning 354277 diluted according to the manufacturer’s COA). Cells were passaged every 5 days using ReLeSR dissociation reagent (Stem Cell Technologies 5872) according to the manufacturer’s protocol. 10 μM of Rho-associated kinase inhibitor (ROCK) Y27632 (EMD 68000) was added to the media for the first 24 hours after passaging. iPS cells were incubated at 37°C in 5% CO2 and maintained at in 2 mL of mTESR media with changes every 48 hours until day of passage.
Han L., Choudhury S., Mich-Basso J.D., Ammanamanchi N., Ganapathy B., Suresh S., Khaladkar M., Singh J., Maehr R., Zuppo D.A., Kim J., Eberwine J.H., Wyman S.K., Wu Y, & Kühn B. (2020). Lamin B2 levels regulate polyploidization of cardiomyocyte nuclei and myocardial regeneration. Developmental cell, 53(1), 42-59.e11.
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5

Generation of Induced Microglial-Like Cells

Cited 1 time
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Human induced pluripotent stem cells (iPSCs) were maintained on Matrigel (BD Biosciences) in complete StemFlex medium (Thermo Fisher #A3349401) supplemented with Pen/Strep. iPSCs were passaged every week using ReLeSR dissociation reagent (StemCell Technologies) and used for hematopoietic stem cell differentiation with STEMdiff Hematopoietic kit (STEMCELL Technologies #05310) followed by differentiation to induced microglial-like cells (iMGLs) using a previously published protocol by (McQuade et al., 2018 (link)). F11349, F12455 and 75.11-IW1A12 (Karch et al., 2019 (link)) iPSC lines were used with confirmed normal karyotyping (WiCell). iMGLs maturation was confirmed with immunostaining for PU.1 and CD11b after 3–5 days of culture in mature microglial medium. Mature iMGLs were transfected with a set of 4 ON-TARGETplus human siRNAs (Horizon Discovery #LQ-010537-00-0002) at 40nM in 6-well plates (~300.000 cells per well in 3–4 ml) using jetPRIME reagent (Polyplus #114–15) for 48 hours. Cells were collected for qPCR analysis as described below using TaqMan Gene Expression Assays (Thermo Fisher) for a set of selected genes in Fig. 1c.
Pimenova A.A., Herbinet M., Gupta I., Machlovi S.I., Bowles K.R., Marcora E, & Goate A.M. (2020). Alzheimer’s-associated PU.1 expression levels regulate microglial inflammatory response. Neurobiology of disease, 148, 105217.
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6

Generation of Induced Microglial-like Cells

Cited 1 time
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Human induced pluripotent stem cells (iPSCs) were maintained on Matrigel (BD Biosciences) in complete mTeSR Plus (STEMCELL Technologies, 100–0276). iPSCs were passaged every 5–6 days using ReLeSR dissociation reagent (STEMCELL Technologies, 05872) and used for hematopoietic stem cell differentiation with STEMdiff Hematopoietic kit (STEMCELL Technologies, 05310) followed by differentiation to induced microglial-like cells (iMGLs) using a previously published protocol [53 (link)]. iPSC lines were confirmed to have a normal karyotype (KaryoStat assay, Thermo Fisher Scientific). iMGLs were maintained and fed with a microglial medium supplemented with three factors (100ng/ml IL34, 50ng/ml TGFβ, 25ng/ml MCSF) for 25 days. On day 25, iMGLs were additionally supplemented with two factors (CX3CL1 and CD200, 100ng/ml each) for an additional three days. Mature iMGLs (day 28) were used for bulk RNA-seq analyses and functional assays.
Podlesny-Drabiniok A., Novikova G., Liu Y., Dunst J., Temizer R., Giannarelli C., Marro S., Kreslavsky T., Marcora E, & Goate A.M. (2023). BHLHE40/41 regulate macrophage/microglia responses associated with Alzheimer’s disease and other disorders of lipid-rich tissues. bioRxiv.
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7

Differentiation of iPSCs to Induced Microglial-like Cells

Cited 1 time
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Human induced pluripotent stem cells (iPSCs) were maintained on Matrigel (BD Biosciences) in complete mTeSR Plus (STEMCELL Technologies, 100-0276). iPSCs were passaged every 5–6 days using ReLeSR dissociation reagent (STEMCELL Technologies, 05872) and used for hematopoietic stem cell differentiation with STEMdiff Hematopoietic kit (STEMCELL Technologies, 05310) followed by differentiation to induced microglial-like cells (iMGLs) using a previously published protocol52 (link). An example of mature iMGLs culture and the expression level of known transcription factors and microglial markers is shown in Supplementary Fig. 8. iPSC lines were confirmed to have a normal karyotype (KaryoStat assay, Thermo Fisher Scientific). iMGLs were maintained and fed with a microglial medium supplemented with three factors (100 ng/ml IL34, 50 ng/ml TGFβ, 25 ng/ml MCSF) for 25 days. On day 25, iMGLs were additionally supplemented with two factors (CX3CL1 and CD200, 100 ng/ml each) for an additional three days. Mature iMGLs (day 28) were used for bulk RNA-seq analyses and functional assays.
Podleśny-Drabiniok A., Novikova G., Liu Y., Dunst J., Temizer R., Giannarelli C., Marro S., Kreslavsky T., Marcora E, & Goate A.M. (2024). BHLHE40/41 regulate microglia and peripheral macrophage responses associated with Alzheimer’s disease and other disorders of lipid-rich tissues. Nature Communications, 15, 2058.
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