Human Pluripotent Stem Cell Functional Identification Kit

Name: Human Pluripotent Stem Cell Functional Identification Kit
Brand: R&D Systems
SKU: SC027B
Price: 686 USD

R&D Systems, Inc. a Bio-Techne Brand

Catalog # SC027B

Citations (50)

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Setting the standard in quality research reagents

Summary for Human Pluripotent Stem Cell Functional Identification Kit

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

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.

Product Specifications for Human Pluripotent Stem Cell Functional Identification Kit

Species

Human

Scientific Data Examples for Human Pluripotent Stem Cell Functional Identification Kit

	Verification of Induced Pluripotent Stem Cell Pluripotency. iPS2 human induced pluripotent stem cells were differentiated to ectoderm, mesoderm, and endoderm using the media supplements included in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027). The kit also contains Goat Anti-Human Otx2 (ectoderm), Goat Anti-Human Brachyury (mesoderm), and Goat Anti-Human SOX17 (endoderm) Antigen Affinity-purified Polyclonal Antibodies for the confirmation of differentiation status. To further evaluate lineage commitment, cells were stained with a Goat Anti-Human SOX1 Antigen Affinity-purified Antibody (Catalog # AF3369), a Goat Anti-Human HAND1 Antigen Affinity-purified Antibody (Catalog # AF3168), and a Goat Anti-Human HNF-3 beta/FoxA2 Antigen Affinity-purified Antibody (Catalog # AF2400). The cells were stained using the NorthernLights^™557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001; red) and the nuclei were counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
	Analysis of Functional Tri-lineage Differentiation using Flow Cytometry. JOY6 human induced pluripotent stem cells were differentiated to ectoderm, mesoderm, and endoderm using the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, Catalog # SC027B). Differentiation was analyzed by flow cytometry using the protocol provided with this kit. Differentiated cells (orange) show increased expression of their respective lineage-specific markers (ectoderm - Otx2, mesoderm - Brachyury, endoderm - SOX17) compared to undifferentiated JOY6 pluripotent stem cells (blue).
	Verification of Human Embryonic Stem Cell Pluripotency. Human embryonic stem cells were differentiated to ectoderm, mesoderm, and endoderm using the media supplements included in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027). The kit also contains Goat Anti-Human Otx2 (ectoderm), Goat Anti-Human Brachyury (mesoderm), and Goat Anti-Human SOX17 (endoderm) Antigen Affinity-purified Polyclonal Antibodies for the confirmation of differentiation status. The cells were stained using the NorthernLights^™557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001; red) and the nuclei were counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
	Verification of Human Induced Pluripotent Stem Cell Pluripotency. Two human induced pluripotent stem cells lines (ADLF1 and FAB2) were differentiated to ectoderm, mesoderm, and endoderm using the media supplements included in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027B). The kit also contains Goat Anti-Human Otx2 (ectoderm), Goat Anti-Human Brachyury (mesoderm), and Goat Anti-Human SOX17 (endoderm) Antigen Affinity-purified Polyclonal Antibodies for the confirmation of differentiation status. The cells were stained using the NorthernLights™ 557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001; red) and the nuclei were counterstained with DAPI (blue).

Preparation & Storage

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.

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

View Graphic Procedure

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.

Add 1.1x10⁵ cells/cm² in 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 (50)

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Citations are publications that use Bio-Techne products. Selected citations for Human Pluripotent Stem Cell Functional Identification Kit include:

Species: Human
Sample Types: Whole Cells

HM Yang et al. (2023), Identification of cell-biologic mechanisms of coronary artery spasm and its ex vivo diagnosis using peripheral blood-derived iPSCs Biomaterials research, 27(1):16.
PMID: 36803875
Species: Human
Sample Types: Whole Cells

S Cho et al. (2022), Heterozygous LMNA mutation-carrying iPSC lines from three cardiac laminopathy patients Stem Cell Research, 59(0):102657.
PMID: 34999423
Species: Human
Sample Types: Whole Cells
Applications: Bioassay

HM Lee et al. (2022), Generation of alphaMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3 using CRISPR/Cas9-based gene targeting Stem Cell Research, 61(0):102779.
PMID: 35427904
Species: Human
Sample Types: Transfected Whole Cells
Applications: ICC

P Mencke et al. (2022), Generation and characterization of a genetic Parkinson's disease-patient derived iPSC line DJ-1-delP (LCSBi008-A) Stem Cell Research, 62(0):102792.
PMID: 35500378
Species: Human
Sample Types: Spheroid
Applications: Bioassay

J Jeong et al. (2022), Elimination of Reprogramming Transgenes Facilitates the Differentiation of Induced Pluripotent Stem Cells into Hepatocyte-like Cells and Hepatic Organoids Biology, 11(4).
PMID: 35453693
Species: Human
Sample Types: Whole Cells

P Mencke et al. (2022), Generation of isogenic control DJ-1-delP GC13 for the genetic Parkinson's disease-patient derived iPSC line DJ-1-delP (LCSBi008-A-1) Stem Cell Research, 62(0):102815.
PMID: 35617842
Species: Human
Sample Types: Transduced Whole Cells

A Kojic et al. (2022), Generation of two induced pluripotent stem cell lines from dilated cardiomyopathy patients carrying heterozygous FLNC mutations Stem Cell Research, 64(0):102928.
PMID: 36194907
Species: Human
Sample Types: Whole Cells

M Alonzo et al. (2022), Characterization of an iPSC line NCHi006-A from a patient with hypoplastic left heart syndrome (HLHS) Stem Cell Research, 64(0):102892.
PMID: 35987121
Species: Human
Sample Types: Whole Cells

J Contreras et al. (2022), Generation of an induced pluripotent stem cell line NCHi003-A from a 11-year-old male with pulmonary atresia with intact ventricular septum (PA-IVS) Stem Cell Research, 64(0):102893.
PMID: 35987120
Species: Human
Sample Types: Whole Cells

J Liu et al. (2022), Generation of a human induced pluripotent stem cell line carrying the TYR c.575C>A (p.Ser192Tyr) and c.1205G>A (p.Arg402Gln) variants in homozygous state using CRISPR-Cas9 genome editing Stem Cell Research, 64(0):102880.
PMID: 35933957
Species: Human
Sample Types: Whole Cells

A Caudal et al. (2022), Generation of human induced pluripotent stem cell lines carrying heterozygous PLN mutation from dilated cardiomyopathy patients Oncogene, 63(0):102855.
PMID: 35853412
Species: Human
Sample Types: Whole Cells

H Lin et al. (2022), Generation and characterization of a human induced pluripotent stem cell (iPSC) line from a patient with congenital heart disease (CHD) Stem Cell Research, 65(0):102958.
PMID: 36343514
Species: Human
Sample Types: Whole Cells

Z Yildirim et al. (2022), Generation of two induced pluripotent stem cell lines from dilated cardiomyopathy patients caused by heterozygous mutations in the HCN4 gene Stem Cell Research, 65(0):102951.
PMID: 36332467
Species: Human
Sample Types: Whole Cells

S Adhicary et al. (2022), Establishment of NCHi009-A, an iPSC line from a patient with hypoplastic left heart syndrome (HLHS) carrying a heterozygous NOTCH1 mutation Stem Cell Research, 66(0):103013.
PMID: 36599283
Species: Human
Sample Types: Whole Cells
Applications: ICC

N Jimenez-Te et al. (2022), Generation of two iPSC lines from long QT syndrome patients carrying SNTA1 variants Stem Cell Research, 66(0):103003.
PMID: 36528013
Species: SC027
Sample Types: Whole Cells
Applications: Bioassay

G Iannello et al. (2021), Generation of the iPSC line CUIMCi003-A derived from a patient with severe early onset obesity Stem Cell Research, 54(0):102432.
PMID: 34214899
Species: Human
Sample Types: Whole Cells

JWS Jahng et al. (2021), Generation of three induced pluripotent stem cell lines, SCVIi003-A, SCVIi004-A, SCVIi005-A, from patients with ARVD/C caused by heterozygous mutations in the PKP2 gene Stem Cell Research, 53(0):102284.
PMID: 33743362
Species: Human
Sample Types: Whole Cells

A Figiel-Dab et al. (2021), Neurogenic and Neuroprotective Potential of Stem/Stromal Cells Derived from Adipose Tissue Cells, 10(6).
PMID: 34208414
Species: Human
Sample Types: Whole Cells

I Boussaad et al. (2021), Integrated, automated maintenance, expansion and differentiation of 2D and 3D patient-derived cellular models for high throughput drug screening Scientific Reports, 11(1):1439.
PMID: 33446877
Species: Human
Sample Types: Whole Cells

N Belbachir et al. (2021), Generation of two induced pluripotent stem cell lines from Brugada syndrome affected patients carrying SCN5A mutations Stem Cell Research, 57(0):102605.
PMID: 34856468
Species: Human
Sample Types: Whole Cells

C Lee et al. (2021), Generation of three iPSC lines from dilated cardiomyopathy patients carrying a pathogenic LMNA variant Stem Cell Research, 59(0):102638.
PMID: 34954454
Species: Human
Sample Types: Whole Cells

SK Sarvestani et al. (2021), Induced organoids derived from patients with ulcerative colitis recapitulate colitic reactivity Nature Communications, 12(1):262.
PMID: 33431859
Species: Human
Sample Types: Whole Cells

G Iannello et al. (2021), Derivation and characterization of the induced pluripotent stem cell line CUIMCi004-A from a patient with a novel frameshift variant in exon 18a of OCRL Stem Cell Research, 59(0):102635.
PMID: 35074682
Species: Human
Sample Types: Whole Cells

X Cao et al. (2021), Generation of three induced pluripotent stem cell lines from hypertrophic cardiomyopathy patients carrying MYH7 mutations Stem Cell Research, 55(0):102455.
PMID: 34352619
Species: Human
Sample Types: Whole Cells

M Machowska et al. (2021), Generation of one control and four iPSCs clones from patients with Emery-Dreifuss muscular dystrophy type 1 Stem Cell Research, 55(0):102487.
PMID: 34419748
Species: Human
Sample Types: Whole Cells

H Zhang et al. (2021), Generation of three induced pluripotent stem cell lines (SCVIi014-A, SCVIi015-A, and SCVIi016-A) from patients with LQT1 caused by heterozygous mutations in the KCNQ1 gene Stem Cell Research, 55(0):102492.
PMID: 34411974
AJ Duarte et al. (2020), Induced pluripotent stem cell line (INSAi002-A) from a Fabry Disease patient hemizygote for the rare p.W287X mutation Stem Cell Res, 45(0):101794.
PMID: 32388441
Species: Human

SB Larsen et al. (2020), Induced pluripotent stem cell line (LCSBi001-A) derived from a patient with Parkinson's disease carrying the p.D620N mutation in VPS35 Stem Cell Res, 45(0):101776.
PMID: 32387898
Species: Human
Sample Types: Whole Cells
Applications: Bioassay

PA Barbuti et al. (2020), Generation of two iPS cell lines (HIHDNDi001-A and HIHDNDi001-B) from a Parkinsonï¿½s disease patient carrying the heterozygous p.A30P mutation in SNCA Stem Cell Res, 48(0):101951.
PMID: 32798915
Species: Human
Sample Types: Whole Cells

S Pagliari et al. (2020), YAP-TEAD1 control of cytoskeleton dynamics and intracellular tension guides human pluripotent stem cell mesoderm specification Cell Death Differ, 0(0).
PMID: 33116297
Species: Human
Sample Types: Whole Cells

Y Zhang et al. (2020), The lncRNA H19 alleviates muscular dystrophy by stabilizing dystrophin Nat Cell Biol, 22(11):1332-1345.
PMID: 33106653
Species: Human
Sample Types: Whole Cells
Applications: Bioassay

P Barbuti et al. (2020), Using High-Content Screening to Generate Single-Cell Gene-Corrected Patient-Derived iPS Clones Reveals Excess Alpha-Synuclein with Familial Parkinson's Disease Point Mutation A30P Cells, 9(9).
PMID: 32927687
Species: Human
Sample Types: Whole Cells

PA Barbuti et al. (2020), Generation of two iPS cell lines (HIHDNDi001-A and HIHDNDi001-B) from a Parkinsonï¿½s disease patient carrying the heterozygous p.A30P mutation in SNCA Stem Cell Res, 48(0):101951.
PMID: 32798915
Species: Human
Sample Types: Whole Cells

S Park et al. (2019), Establishment of PITX3-mCherry knock-in reporter human embryonic stem cell line (WAe009-A-23) Stem Cell Res, 39(0):101499.
PMID: 31352199
Species: Human
Sample Types: Whole Cells

S Kanton et al. (2019), Organoid single-cell genomic atlas uncovers human-specific features of brain development Nature, 574(7778):418-422.
PMID: 31619793
Species: Human
Sample Types: Whole Cells

C Lu et al. (2019), Generation of a knock-in MAP2-tdTomato reporter human embryonic stem cell line with inducible expression of NEUROG2/1 (NYGCe001-A) Stem Cell Res, 41(0):101643.
PMID: 31707212
Species: Human
Sample Types: Whole Cells

AJ Duarte et al. (2019), Induced pluripotent stem cell line (INSAi001-A) from a Gaucher disease type 3 patient compound heterozygote for mutations in the GBA1 gene Stem Cell Res, 41(0):101595.
PMID: 31678773
Species: Human
Sample Types: Whole Cells

AL Gagne et al. (2018), Generation of a human Juvenile myelomonocytic leukemia iPSC line, CHOPi001-A, with a mutation in CBL Stem Cell Res, 31(0):157-160.
PMID: 30096712
Species: Human
Sample Types: Whole Cells

Y Nakashima et al. (2018), Atorvastatin Inhibits the HIF1?-PPAR Axis, Which Is Essential for Maintaining the Function of Human Induced Pluripotent Stem Cells Mol. Ther., 0(0).
PMID: 29929789
Species: Human
Sample Types: Whole Cells

JA Maguire et al. (2018), 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 Stem Cell Res, 34(0):101361.
PMID: 30611021
Species: Human
Sample Types: Whole Cells

Y Nam et al. (2017), Cord blood cell-derived iPSCs as a new candidate for chondrogenic differentiation and cartilage regeneration Stem Cell Res Ther, 8(1):16.
PMID: 28129782
Species: Human
Sample Types: Whole Cells

T Shinozawa et al. (2017), Recapitulation of Clinical Individual Susceptibility to Drug-Induced QT Prolongation in Healthy Subjects Using iPSC-Derived Cardiomyocytes Stem Cell Reports, 0(0).
PMID: 28111276
Species: Human
Sample Types: Whole Cells
Applications: ICC

Glenda M Halliday (2016), Toll-like receptor 2 is increased in neurons in Parkinson's disease brain and may contribute to alpha-synuclein pathology Acta Neuropathol., 0(0).
PMID: 27888296
Species: Human
Sample Types: Whole Cells

Claudia Altomare (2016), Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade Europace, 18(0):iv67-iv76.
PMID: 28011833
Species: Human
Sample Types: Whole Cells
Applications: Bioassay

(2016), 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(0):1329459.
PMID: 27579041
Species: Human
Sample Types: Whole Cells
Applications: Bioassay

Krueger et al. (2013), Cholesterol-secreting and statin-responsive hepatocytes from human ES and iPS cells to model hepatic involvement in cardiovascular health. PLoS ONE, 8(7):e67296.
PMID: 23874411
Species: Bovine
Sample Types: Whole Cells
Applications: Bioassay

Furusawa et al. (2013), Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses. Biol Reprod, 89(2):28.
PMID: 23782837
Species: Human
Sample Types: Whole Cells
Applications: ICC

Szablowska-Gadomska et al. (2012), Treatment with small molecules is an important milestone towards the induction of pluripotency in neural stem cells derived from human cord blood. Acta Neurobiol Exp (Wars), 72(4):337-50.
PMID: 23377265
Chen B et al. (0), Inducible overexpression of RUNX1b/c in human embryonic stem cells blocks early hematopoiesis from mesoderm. J Mol Cell Bio9(4):262-273.
PMID: 28992293
Species: Human
Sample Types: Whole Cells

Deshpande A et al. (0), Cellular Phenotypes in Human iPSC-Derived Neurons from a Genetic Model of Autism Spectrum Disorder. Cell Re21(10):2678-2687.
PMID: 29212016

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