Human Pluripotent Stem Cell Functional Identification Kit

Catalog # Availability Size / Price Qty
SC027B
Human Pluripotent Stem Cell Functional Identification Kit
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Citations (19)
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Human Pluripotent Stem Cell Functional Identification Kit Summary

Kit Summary

To verify pluripotency in human stem cells by in vitro functional differentiation.

Key Benefits

  • Verifies the pluripotency of your starting population
  • Only takes 5 days
  • Reduces experimental variation
 
Stemness vs. Pluripotency
 

 

Why functionally verify human stem cell pluripotency in vitro?

To determine if a cell is truly a pluripotent stem cell, it is important to verify its ability to differentiate into each of the three germ layers.

The teratoma assay is a standard way to assess pluripotency, but this method requires expensive animal models and is time-consuming, often requiring 6 to 10 weeks before pluripotency status can be determined. Similarly, embryoid body assays are time-consuming and rely on random differentiation.

  • Uses fully defined supplements to drive reproducible differentiation.
  • Provides results in 5 days to save time and reagents.
  • Avoids tissue embedding and sectioning procedures.
  • Can verify the ability to differentiate into 3 germ layers without the use of an animal model.
  • Verifies a healthy, pluripotent starting stem cell population to increase consistency between studies and reduce unwanted experimental variability.

 

Kit Contents

This kit contains the following specially formulated media supplements and growth factors to drive pluripotent stem cell differentiation and a marker to characterize each of the three germ layer cell types.

  • Differentiation Base Media Supplement (50X)
  • Ectoderm, Mesoderm, and Endoderm Differentiation Supplements
  • Ectoderm Marker: Goat Anti-Human Otx2 Antigen Affinity-purified Polyclonal Antibody
  • Mesoderm Marker: Goat Anti-Human Brachyury Antigen Affinity-purified Polyclonal AntibodyV
  • Endoderm Marker: Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody

The quantity of each component in this kit is sufficient to make 200 mL of media for differentiation. This is enough media for the differentiation of one 24-well plate of each cell type.

The acute and chronic effects of over-exposure to the reagents in this kit are unknown. Safe laboratory handling procedures should be followed and protective clothing should be worn when handling kit reagents.

 

Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of pre-implantation embryos. Induced pluripotent stem (iPS) cells can be generated by somatic cell reprogramming following the exogenous expression of specific transcription factors (Oct-3/4, KLF4, SOX2, and c-Myc). These cell types are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity to differentiate into a wide variety of somatic cells. In this capacity, pluripotent stem cells have widespread clinical potential for the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders.

R&D Systems offers a wide range of products to support pluripotent stem cell culture and differentiation. Mouse embryonic fibroblasts may be used to maintain and expand pluripotent stem cells in an undifferentiated state. We also offer defined culture media, which are specifically optimized for use with human or rodent pluripotent stem cells. In addition, R&D Systems offers a variety of products to assess differentiation status and identify specific stem cell types of interest, including panels of marker antibodies, primer pairs, multi-color flow cytometry kits, and specialized verification kits.

Specifications

Shipping Conditions
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Species
Human

Product Datasheets

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, the pluripotency status of human stem cells is verified using the following in vitro differentiation procedure:

  • Culture pluripotent cells of interest
  • Induce endoderm, mesoderm, and ectoderm differentiation with media supplements
  • Evaluate differentiation using germ layer markers and fluorescent ICC
 

 

Kit Components

Reagents supplied in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027B):

  • Differentiation Base Media Supplement (50X)
  • Ectoderm Differentiation Supplement
  • Mesoderm Differentiation Supplement
  • Endoderm Differentiation Supplement I
  • Endoderm Differentiation Supplement II
  • Ectoderm Marker: Goat Anti-Human Otx2 Antigen Affinity-purified Polyclonal Antibody
  • Mesoderm Marker: Goat Anti-Human Brachyury Antigen Affinity-purified Polyclonal Antibody
  • Endoderm Marker: Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody
     

    Note: The quantity of each component in this kit is sufficient to make 200 mL of medium for differentiation. This is enough medium for the differentiation of one 24-well plate for each lineage.

The quantity of each component in this kit is sufficient to differentiate two 24-well plates, or an equivalent surface area, of pluripotent stem cells into hepatocyte-like cells.

 

Other Supplies Required

Reagents

  • RPMI
  • BSA, very low endotoxin
  • D-MEM/F-12 (1X)
  • GlutaMAX™ (Invitrogen or equivalent)
  • Penicillin-Streptomycin
  • Phosphate Buffered Saline (PBS)
  • Trypan Blue Solution
  • MEF Conditioned Media (Catalog # AR005 or equivalent)
  • StemXVivo Culture Matrix (100X) (Catalog # CCM013), Cultrex® PathClear® BME Reduced Growth Factor Basement Membrane Extract (Catalog # 3433-005-01), or equivalent
  • Recombinant Human FGF basic (Tissue culture grade; Catalog # 4114-TC or equivalent)
  • Accutase® (Innovative Cell Technologies or equivalent)

Materials

  • Human pluripotent stem cells
  • 24-well culture plates
  • 15 mL centrifuge tubes
  • 50 mL centrifuge tubes
  • 0.2 μm syringe filter
  • 10 mL syringe
  • Pipettes and pipette tips
  • Serological pipettes

Equipment

  • 37 °C and 5% CO2 incubator
  • Centrifuge
  • Hemocytometer
  • Inverted microscope
  • 37 °C water bath

 

Procedure Overview

This protocol is designed for BG01V human embryonic stem (hES) cells and iPS2 human induced pluripotent stem (iPS) cells grown in Mouse Embryonic Fibroblast (MEF) Conditioned Media (Catalog # AR005). If using different cell lines or growth media, this protocol may need to be optimized.

Note: The quality of the human pluripotent cells used in the differentiation is imperative. Use of suboptimal quality or very high passage pluripotent cells can result in decreased differentiation efficiency and/or increased cell death.

Undifferentiated Cell Preparation

Add 1:100 Culture Matrix or Cultrex BME in sterile PBS.

Culture Matrix or Cultrex BME in sterile PBS

Add 1.1x105 cells/cm2 in MEF Conditioned Media containing 4 ng/mL FGF Basic.

MEF Conditioned Media containing 4 ng/mL FGF Basic
 

Differentiation Procedures

 

Ectoderm
Differentiation

Mesoderm
Differentiation

Endoderm
Differentiation

Day 1 Replace media with Ectoderm Differentiation Media. Replace media with
Mesoderm Differentiation Media.
Replace media with Endoderm Differentiation Media.
Day 2 Repeat Repeat media change 12-16 hours later.
ICC detection of Brachyury (24-36 hours after initial differentiation)
16-24 hours later, Replace media with Endoderm Differentiation Media II.
Day 3 Repeat   Replace media with Endoderm Differentiation Media II.
Day 4 ICC detection of Otx2.   ICC detection of SOX17.

Citations for Human Pluripotent Stem Cell Functional Identification Kit

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

19 Citations: Showing 1 - 10
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  1. Induced pluripotent stem cell line (LCSBi001-A) derived from a patient with Parkinson's disease carrying the p.D620N mutation in VPS35
    Authors: SB Larsen, Z Hanss, G Cruciani, F Massart, PA Barbuti, G Mellick, R Krüger
    Stem Cell Res, 2020;45(0):101776.  2020
  2. Induced pluripotent stem cell line (INSAi002-A) from a Fabry Disease patient hemizygote for the rare p.W287X mutation
    Authors: AJ Duarte, D Ribeiro, R Santos, L Moreira, J Bragança, O Amaral
    Stem Cell Res, 2020;45(0):101794.  2020
  3. Induced pluripotent stem cell line (INSAi001-A) from a Gaucher disease type 3 patient compound heterozygote for mutations in the GBA1 gene
    Authors: AJ Duarte, D Ribeiro, R Santos, L Moreira, J Bragança, O Amaral
    Stem Cell Res, 2019;41(0):101595.  2019
  4. Generation of a knock-in MAP2-tdTomato reporter human embryonic stem cell line with inducible expression of NEUROG2/1 (NYGCe001-A)
    Authors: C Lu, NE Sanjana
    Stem Cell Res, 2019;41(0):101643.  2019
  5. Organoid single-cell genomic atlas uncovers human-specific features of brain development
    Authors: S Kanton, MJ Boyle, Z He, M Santel, A Weigert, F Sanchís-Ca, P Guijarro, L Sidow, JS Fleck, D Han, Z Qian, M Heide, WB Huttner, P Khaitovich, S Pääbo, B Treutlein, JG Camp
    Nature, 2019;574(7778):418-422.  2019
  6. Establishment of PITX3-mCherry knock-in reporter human embryonic stem cell line (WAe009-A-23)
    Authors: S Park, JE Yoo, DR Lee, J Jang, MS Cho, DS Kim, DW Kim
    Stem Cell Res, 2019;39(0):101499.  2019
  7. Generation of a human Juvenile myelomonocytic leukemia iPSC line, CHOPi001-A, with a mutation in CBL
    Authors: AL Gagne, JA Maguire, S Gandre-Bab, ST Chou, SK Tasian, ML Loh, MJ Weiss, P Gadue, DL French
    Stem Cell Res, 2018;31(0):157-160.  2018
  8. Atorvastatin Inhibits the HIF1?-PPAR Axis, Which Is Essential for Maintaining the Function of Human Induced Pluripotent Stem Cells
    Authors: Y Nakashima, C Miyagi-Shi, H Noguchi, T Omasa
    Mol. Ther., 2018;0(0):.  2018
  9. Generation of Spinocerebellar Ataxia Type 2 induced pluripotent stem cell lines, CHOPi002-A and CHOPi003-A, from patients with abnormal CAG repeats in the coding region of the ATXN2 gene
    Authors: JA Maguire, AL Gagne, P Gonzalez-A, BL Davidson, V Shakkottai, P Gadue, DL French
    Stem Cell Res, 2018;34(0):101361.  2018
  10. Recapitulation of Clinical Individual Susceptibility to Drug-Induced QT Prolongation in Healthy Subjects Using iPSC-Derived Cardiomyocytes
    Authors: T Shinozawa, K Nakamura, M Shoji, M Morita, M Kimura, H Furukawa, H Ueda, M Shiramoto, K Matsuguma, Y Kaji, I Ikushima, M Yono, SY Liou, H Nagai, A Nakanishi, K Yamamoto, S Izumo
    Stem Cell Reports, 2017;0(0):.  2017
  11. Cord blood cell-derived iPSCs as a new candidate for chondrogenic differentiation and cartilage regeneration
    Authors: Y Nam, YA Rim, SM Jung, JH Ju
    Stem Cell Res Ther, 2017;8(1):16.  2017
  12. The Generation of Human Induced Pluripotent Stem Cells from Blood Cells: An Efficient Protocol Using Serial Plating of Reprogrammed Cells by Centrifugation
    Stem Cells Int, 2016;2016(0):1329459.  2016
  13. Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade
    Authors: Claudia Altomare
    Europace, 2016;18(0):iv67-iv76.  2016
  14. Toll-like receptor 2 is increased in neurons in Parkinson's disease brain and may contribute to alpha-synuclein pathology
    Authors: Glenda M Halliday
    Acta Neuropathol., 2016;0(0):.  2016
  15. Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.
    Authors: Furusawa, Tadashi, Ohkoshi, Katsuhir, Kimura, Koji, Matsuyama, Shuichi, Akagi, Satoshi, Kaneda, Masahiro, Ikeda, Mitsumi, Hosoe, Misa, Kizaki, Keiichir, Tokunaga, Tomoyuki
    Biol Reprod, 2013;89(2):28.  2013
  16. Cholesterol-secreting and statin-responsive hepatocytes from human ES and iPS cells to model hepatic involvement in cardiovascular health.
    Authors: Krueger, Winfried, Tanasijevic, Borko, Barber, Vanessa, Flamier, Anthony, Gu, Xinsheng, Manautou, Jose, Rasmussen, Theodore
    PLoS ONE, 2013;8(7):e67296.  2013
  17. Treatment with small molecules is an important milestone towards the induction of pluripotency in neural stem cells derived from human cord blood.
    Authors: Szablowska-Gadomska, Ilona, Sypecka, Joanna, Zayat, Valery, Podobinska, Martyna, Pastwinska, Anna, Pienkowska-Grela, Barbara, Buzanska, Leonora
    Acta Neurobiol Exp (Wars), 2012;72(4):337-50.  2012
  18. Cellular Phenotypes in Human iPSC-Derived Neurons from a Genetic Model of Autism Spectrum Disorder.
    Authors: Deshpande A, Yadav S, Dao D, Wu Z, Hokanson K, Cahill M, Wiita A, Jan Y, Ullian E, Weiss L
    Cell Rep, 0;21(10):2678-2687.  0
  19. Inducible overexpression of RUNX1b/c in human embryonic stem cells blocks early hematopoiesis from mesoderm.
    Authors: Chen B, Teng J, Liu H, Pan X, Zhou Y, Huang S, Lai M, Bian G, Mao B, Sun W, Zhou Q, Yang S, Nakahata T, Ma F
    J Mol Cell Biol, 0;9(4):262-273.  0

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