ES/iPS cells

1 Introduction

Pluripotent stem cells (PSCs) are self-renewing stem cells with the ability to differentiate into all cell types of the embryo. They can give rise to all cell types derived from the germ layers ectoderm, mesoderm, and endoderm, a feature known as pluripotency.

Their enormous developmental potential makes PSCs prime candidates for applications in regenerative medicine, where lineage-specific progeny cells are used to replace degenerated or diseased cells and tissues (PMID: 24434846). PSCs are also a powerful tool for studying human diseases and development. PSC differentiation in vitro can partially recapitulate human development in vivo. Comparing PSCs from healthy and diseased donors can provide insight into the pathology of human diseases and, in combination with modern genome editing techniques, opens the possibility of modeling disease-specific lesions. Finally, PSCs have opened new avenues for drug and toxicity screening (PMID: 23746976).

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2 Pluripotent stem cell subtypes

Based on their origin, PSCs can be classified as human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells.

Human ES cells are derived from the inner cell mass of a developing human embryo and were isolated for the first time in 1998 by James Thomson (PMID: 9804556). Use of ES cells is controversial because their derivation involves the destruction of a human embryo, and many countries have imposed restrictions on their de novo generation and use, such as the regulation of stem cell research in Europe.  

iPS cells are derived from somatic cells via reprogramming. Due to their somatic origin, their use is viewed as ethically less problematic. For details about reprogramming, see the section below: Somatic cell reprogramming.

Both cell types grow as cell lines that form characteristic adherent colonies. In terms of morphology, marker expression, developmental potential, and experimental handling, ES and iPS cells are essentially indistinguishable.

3 Miltenyi Biotec application protocols for pluripotent stem cells

Miltenyi Biotec has created dedicated application protocols to maintain and cryopreserve PSC lines.

4 Somatic cell reprogramming

In 2006, Takahashi and Yamanaka reported the reprogramming of somatic cells to a pluripotent state using defined transcription factors (PMID: 16904174). iPS cells have since become an invaluable tool for strategies in basic developmental research, disease modeling, drug discovery, and regenerative medicine.

Several methodologies have been developed for the de novo generation of iPS cells (PMID: 26883003). The webinar in the related resources panel to the right offers an overview of reprogramming methods.

Miltenyi Biotec offers clinically relevant reprogramming tools for iPS cell generation based on non-integrative mRNA transfection technology. mRNA reprogramming has the highest efficiency among all current technologies (>1%) and is the safest and fastest method for the de novo generation of iPS cell lines (PMID: 25437882). Somatic cell types amenable to reprogramming with this technique include human fibroblasts, endothelial progenitors, and renal epithelial cells.

4.1 Establishment of a fibroblast culture for reprogramming

At a glance: Kits and reagents for the generation of a fibroblast culture

Use Comments Product
Tissue dissociationWhole Skin Dissociation Kit, human

Establishing high-quality cultures of proliferating fibroblasts is key to the success of reprogramming experiments. The Whole Skin Dissociation Kit, human in combination with the gentleMACS™ Octo Dissociator with Heaters enables gentle and efficient dissociation of human skin biopsies into single-cell suspensions and the subsequent generation of fibroblast cultures from patient samples. Unlike traditional outgrowth cultures, starting with a single-cell suspension results in consistent monolayer cultures and yields enough fibroblasts for reprogramming within 5–8 days after plating, depending on biopsy size and cell number required for the planned reprogramming method.

4.2 mRNA reprogramming of fibroblasts

mRNA reprogramming technology outperforms other methods in conversion efficiency and kinetics and offers a significant improvement for fast, safe, and effective reprogramming of mature human cells. This method also eliminates the risk of genomic integration and mutagenesis inherent to DNA- and virus-based technologies (PMID: 20888316, 25437882).

5 Magnetic separation of reprogrammed iPS cells

Magnetic cell separation offers an effective and fast means of isolating reprogrammed iPS cells to establish new iPS cell lines. Traditionally, iPS cells are isolated by picking individual colonies, which assumes that each colony represents a single reprogramming event and enables derivation of a clonal, genetically homogeneous cell population. Live cell staining antibodies for the pluripotency markers TRA-1-60 or TRA-1-81 can assist identifying and picking reprogrammed colonies, but this method is nonetheless labor intensive and requires experience in identifying reprogrammed colonies and transferring them to a new culture dish.

With magnetic cell separation, reprogrammed cells can be isolated in bulk (PMID: 23734247). Multiple cell lines can be processed simultaneously, generating large amounts of cells in a short time, and the process can be automated. Furthermore, bulk cells can be subjected to subcloning, if clonal cultures are required. For details on MACS® Cell Separation Technology, see the MACS Handbook chapter  Magnetic cell separation.

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5.1 Isolation of reprogrammed iPS cells

At a glance: Kits and reagents for the separation of reprogrammed iPS cells

Starting material Isolation strategy Comments Automation  Product
Single-cell suspension of cell culturePositive selection of target cellsYesAnti-TRA-1-60 MicroBeads, human
Single-cell suspension of cell culturePositive selection of target cellsYesAnti-SSEA-4 MicroBeads, human
Single-cell suspension of cell cultureDepletion of non-target cellsRemoval of fibroblastsYesAnti-Fibroblast MicroBeads, human

MACS Cell Separation Technology offers an easy way to purify reprogrammed iPS cells based on pluripotency markers such as TRA-1-60 or SSEA-4 (PMID: 21637193, 26281015). Alternatively, isolation can also be achieved by depleting unwanted fibroblasts (PMID: 26237226) using the Anti-Fibroblast MicroBeads, human.

An article describing the magnetic isolation iPS cells after reprogramming can be downloaded from the Related resources panel to the right.

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6 Maintenance of pluripotent stem cell lines

Propagation of PSCs requires special attention and qualified reagents to maintain their pristine features. Different basic cultivation principles have been developed over time and have different advantages and disadvantages.

A webinar describing the different cultivation systems for PSCs can be accessed from the Related resources panel to the right.

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Cultivation systems for PSCs

6.1 Feeder-based culture

At a glance: Kits and reagents for the removal of feeder cells from cocultures with PSCs

Use Comments Product
Feeder cell depletionFeeder Removal MicroBeads, mouse
Feeder cell depletionAnti-Fibroblast MicroBeads, human

First approaches to human ES cell derivation were carried out in the presence of supportive feeder cells (PMID: 9804556), such as mitotically inactivated primary mouse embryonic fibroblasts (MEFs), as well as several fibroblast cell lines (NIH/3T3, STO). These cells support the growth of both mouse and human PSCs through the secretion of cytokines and extracellular matrix molecules (PMID: 22760161, 22099024). Optimal maintenance of pluripotency feeder-based cultures requires further supplementation with low amounts of human FGF-2 and human serum or serum replacement. Feeder cell coculture became the gold standard to work with human PSCs and is still widely used. However, feeder-based coculture requires separating PSCs from feeder cells prior to further analysis or experimentation.

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Strategies for the removal of feeder cells. (A) Mouse embryonic fibroblasts (MEFs) adhere more readily to uncoated tissue culture plates than PSCs, enabling separation of the two cell types. (B) PSCs can be weaned off feeder cells over several passages. (C) Finally, feeder cells are easily and efficiently removed from single-cell suspensions using MACS Technology.

The presence of feeder-cells can interfere with sensitive downstream applications, such as differentiation or gene expression profiling. Removal of feeder cells can be done by leveraging the preferential adherence of mouse embryonic fibroblasts (MEFs) to uncoated tissue culture plates, by weaning off the feeder cells over several passages, or by magnetic cell separation. The first two protocols are time-consuming, do not remove all feeder cells, and lead to considerable loss of pluripotent cells. The weaning method can also cause feeder-adapted PSCs to enter a crisis characterized by a high degree of spontaneous differentiation and impaired lineage differentiation. In contrast, MACS MicroBead Technology is a simple, yet efficient method for feeder cell removal. The Feeder Removal MicroBeads, mouse enable depletion of MEFs in 20 minutes. For removal of human feeder cells, Anti-Fibroblast MicroBeads, human are the tool of choice.

Feeder Removal MicroBeads, mouse
Before separation
After separation

Depletion of feeder cells by magnetic cell separation. Mouse embryonic fibroblasts (MEFs) were depleted from a coculture with mouse ES cells using Feeder Removal MicroBeads, mouse, an LS Column, and a MidiMACS™ Separator. Flow cytometric analysis with CD15 (SSEA-1)-APC and Anti-Feeder-PE on a MACSQuant® Analyzer indicates that a starting population of 10% MEFs was completely removed. The purity of the enriched ES cells was about 99.9%.

6.2 Feeder-free culture

At a glance: Kits and reagents for feeder-free culture of PSCs

Use Comments Product
MediaResearch grade StemMACS iPS-Brew XF, human
MediaGMP gradeiPS-Brew GMP Basal Medium
SupplementFor use with iPS-Brew GMP Basal MediumiPS-Brew GMP Supplement R

As PSCs move closer to clinical application, alternatives to coculture with feeder cells eliminate the qualification and risk assessment required for feeder cells as additional components of a culture system. The first culture system for feeder-free propagation of PSCs was described in 2006 (PMID: 16862139), and has since been refined by reducing the number of components (PMID: 21478862).

Miltenyi Biotec developed a media formulation that combines robustness and reproducibility with a reduced number of components, and is thus suitable for GMP-grade production. The research-grade StemMACS™ iPS-Brew XF and GMP-grade iPS-Brew GMP Medium enable a seamless transition from basic research to manufacturing for cell therapy applications.

Feeder-free culture systems replace the feeder layer with a coating matrix that facilitates cell attachment. Various extracellular matrices are used, including Matrigel® and its derivatives, which have components of animal origin, and vitronectin or Laminin-521, which are recombinant, chemically defined matrices (PMID: 24463987). StemMACS iPS-Brew XF can be combined with any commonly used matrix. However, use of Laminin-521 is recommended because it maintains the xeno-free culture system and provides superior attachment.

Culture protocols initially called for daily media replacement. Especially with the first generation of feeder-free PSC culture media, any deviation from standard protocol could lead to increased spontaneous cell differentiation, slower growth rates, or down-regulation of pluripotency markers. Initial passages often looked fine, but changes became obvious during long-term culture with reduced feeding. Newer media formulations enable a greater flexibility in feeding schedules. This feature is frequently attributed to the substitution of native FGF-2 with thermostabilized versions of the growth factor. Miltenyi Biotec's in-house data indicate, however, that the overall media composition and ability to provide a balanced and robust source of energy, nitrogen, amino acids, and salts is at least if not more important in affording that flexibility. StemMACS iPS-Brew XF enables flexible feeding regimes, including the possibility of skipping one or two feeding days, and has been tested for maintenance of proliferation rate, pluripotent morphology and surface markers over five passages. Results of this analysis can be downloaded from the Related resources panel to the right.

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PSC lines cultured in StemMACS iPS-Brew XF have the typical pluripotent morphology and surface phenotype.
 

6.3 Splitting pluripotent stem cell cultures

At a glance: Kits and reagents for passaging PSCs

Use Comments Product
Routine passagingOptimized for use with StemMACS iPS-Brew XFStemMACS Passaging Solution XF
ROCK inhibitionStemMACS Y27632
ROCK inhibitionStemMACS Thiazovivin

Different PSC lines have different growth kinetics. Therefore, the optimal time and ratio for passaging must be determined individually for each cell line. Generally, it is good practice to keep cultures in a subconfluent state, where colonies are  separated visibly. Cultures should be split before they reach confluency to avoid fusion of colonies and a rapid drop of growth factor levels.

A video showing various passaging techniques for PSCs can be accessed from the Related resources panel to the right.

Detachment of human PSCs must be gentle to maintain maximum viability. StemMACS Passaging Solution XF is designed to minimize cell culture manipulation, eliminating lengthy inactivation, dilution, and centrifugation steps. The ready-to-use formulation facilitates a reproducible and standardized splitting procedure. 

Human PSCs show decreased survival and attachment when colonies are dissociated into single cells or small cell clusters. ROCK inhibitors such as StemMACS Y-27632 or StemMACS Thiazovivin counter this effect and increase survival during passaging and thawing after cryopreservation (PMID: 17529971, 20406903).
 

6.4 Cryopreservation of pluripotent stem cells

At a glance: Kits and reagents for the cryopreservation of PSCs

Use Comments Product
Cell detachmentOptimized for use with StemMACS iPS-Brew XFStemMACS Passaging Solution XF
Cryopreservation solutionStemMACS Cryo-Brew

Optimal storage of PSC lines is critical to maintaining a pool of stable lines, preserved at early passage number. StemMACS Cryo-Brew is a chemically defined media formulation that preserves PSCs with >90% viability and enables rapid culture initiation after thawing. Adding a ROCK inhibitor to the culture medium after thawing provides optimal attachment and survival because cells recover as single cells or small cell clusters (see Splitting pluripotent stem cells cultures).

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Recovery of cryopreserved human PSCs. Cell recovery after thawing is high for PSCs cryopreserved in StemMACS Cryo-Brew. 

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Recovery and viability of cryopreserved human PSCs.  The doubling time of PSCs cryopreserved in StemMACS Cryo-Brew is short compared to cells preserved in conventional freezing medium.

StemMACS Cryo-Brew is also suited for the storage of other cell types, including PSC-derived neural and cardiac progenitors, hematopoietic stem cells, mesenchymal stem cells, and T cells. A scientific poster on the efficiency of StemMACS Cryo-Brew in preserving various cell types can be downloaded from the Related resources panel to the right.

6.5 Magnetic separation of cultured pluripotent stem cells

At a glance: Kits and reagents for the separation of PSCs

Starting material Isolation strategy Comments Automation  Product
Single-cell suspension of cell culturePositive selection of target cellsYesAnti-TRA-1-60 MicroBead Kit, human
Single-cell suspension of cell culturePositive selection of target cellsYesAnti-SSEA-4 MicroBeads, human

If cultured under suboptimal conditions (e.g. insufficient levels of FGF-2) or exposed to environmental stress (e.g. change of culture media or attachment matrix, overconfluent cultures), PSCs may undergo unwanted, spontaneous differentiation. MACS MicroBeads, such as the Anti-TRA-1-60 MicroBead Kit, human and the Anti-SSEA-4 MicroBeads, human, efficiently remove differentiated cells to generate a pure homogeneous population of PSCs for subsequent culture or differentiation.

Anti-TRA-1-60 MicroBeads, human
Before separation
After separation

Efficient separation of undifferentiated PSCs. Pluripotent (TRA-1-60+) iPSCs were isolated from cultures containing spontaneously differentiated iPSCs grown on mouse embryonic feeder cells using Anti-TRA-1-60 MicroBeads, an MS Column, and a MiniMACS™ Separator. Cells were fluorescently stained with Anti-TRA-1-60-PE and Anti-Feeder-APC after separation and analyzed by flow cytometry using a MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

While magnetic separation may help overcome problems caused by temporarily suboptimal culture, it is essential to place the separated cells back into an appropriate culture environment. If the extent of spontaneous differentiation is high, purification should be repeated during the next split. Also note that some PSC lines show an inherently high tendency to spontaneously differentiate. If such lines must be maintained and cannot be discarded within the scope of a research project, immunomagnetic separation can be used to clean up cultures prior to downstream experiments. However, separation may not lead to homogeneous long-term cultures in these cases.

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7 Characterization of pluripotent stem cells by flow cytometry

Human PSC lines can vary significantly with respect to their growth and differentiation characteristics. The genetic and epigenetic basis of this heterogeneity is still poorly understood and the subject of ongoing research (PMID: 23673969).

Thorough and comprehensive characterization is therefore mandatory when establishing new ES or iPS cell lines and good practice during propagation of established lines. A typical basic characterization includes assessment of morphology, extra- and intracellular marker expression, pluripotent differentiation potential, and karyotyping.

7.1 Extra- and intracellular marker expression

At a glance: Markers for the detection of undifferentiated human PSCs

Cell surfaceIntracellular
TRA-1-60Oct-4
TRA-1-81Sox-2
SSEA-3Nanog
SSEA-4
SSEA-5
CD326 (EpCAM)

In addition to antibodies against pluripotency markers, antibodies that detect signs of early differentiation (e.g. Anti-SSEA-1 Antibodies) or the presence of feeder cells (e.g., Anti-Fibroblast or Anti-Feeder Antibodies) can complement the analysis. Especially the detection of intracellular antigens has often been hampered by high background signals when conventional mono- or polyclonal antibodies were used for flow cytometric analysis. In contrast, Miltenyi Biotec's REAfinity™ Recombinant Antibodies ensure highly specific detection with low background signal. For details, see chapter Cell analysis – reagents.

The pluripotency and differentiation status of PSC cultures can be monitored by immunofluorescence microscopy or flow cytometry. The latter allows quantitative evaluation of culture composition and comparison of different cell lines and data points. A detailed protocol for flow cytometric quality control of PSC cultures with up to six markers can be downloaded from the Related resources panel to the right.
 

7.2 Assessment of pluripotent differentiation potential

At a glance: Kits and reagents for the assessment of PSC differentiation potential

UseCommentsProduct
Directed differentiationDifferentiation of PSCs into ectoderm, mesoderm, and endodermStemMACS Trilineage Differentiation Kit, human
Flow cytometric analysisQuantitative analysis of differentiated cells using REAfinity AntibodiesCD144 (VE-Cadherin)-FITC, human (clone: REA199)
Flow cytometric analysisQuantitative analysis of differentiated cells using REAfinity AntibodiesCD140b-APC, human (clone: REA363)
Flow cytometric analysisQuantitative analysis of differentiated cells using REAfinity AntibodiesCD184 (CXCR4)-APC, human (clone: REA649)
Flow cytometric analysisQuantitative analysis of differentiated cells using REAfinity AntibodiesAnti-Sox17-Vio515, human (clone: REA701)
Flow cytometric analysisQuantitative analysis of differentiated cells using REAfinity AntibodiesAnti-PAX-6-APC, human (clone: REA507)
Flow cytometric analysisQuantitative analysis of differentiated cells using REAfinity AntibodiesAnti-Sox2-FITC, human and mouse (clone: REA320)

Traditional assays to assess the differentiation potential of human PSCs include teratoma formation in mice and embryoid body formation in low-attachment cell culture vessels. Both assays are time consuming and difficult to reproduce and quantify.

An alternative is directed differentiation into derivatives of the ectodermal, mesodermal, and endodermal lineages. However, to enable the comparison of different cell lines, the same differentiation procedure must be used for all cultures, with highly consistent protocols and reagents. Miltenyi Biotec has developed the StemMACS Trilineage Differentiation Kit, human, which provides standardized, ready-to-use media for differentiation into ectoderm, mesoderm, and endoderm in seven days. Combined with flow cytometry analysis of the resulting differentiated cells, this kit enables a quantitative comparison of the pluripotency of different PSC lines.

A scientific poster describing the quantitative assessment of PSC differentiation potential, as well as a detailed protocol for the analysis of differentiated samples by flow cytometry can be downloaded from the Related resources panel to the right.

8 Differentiation of ES/iPS cells

8.1 Formation and dissociation of embryoid bodies

At a glance: Kits and reagents for the dissociation of embryoid bodies

When deprived of pluripotent growth factors, PSCs will undergo spontaneous differentiation and give rise to a mix of endodermal, ectodermal, and mesodermal cell lineages to form embryoid bodies (EB). The Embryoid Body Dissocation Kit, human and mouse can be combined with the gentleMACS Dissociator to dissociate these spheroids into single cells in a reproducible manner. The enzyme cocktail contained in the kit preserves the majority of cell surface epitopes, allowing a reliable analysis or separation of differentiated target cells from the heterogeneous single-cell suspension.

8.2 Further differentiation of ES and iPS cells

A variety of differentiation procedures have been developed to maximize the yield of the desired differentiated target cells. The following table provides an overview of differentiation protocols known to work with feeder-free iPSC cultures using StemMACS iPS-Brew XF.


 

 Reagents to stimulate differentiation 
Target cell typeSmall moleculesCytokinesReference (PMID)
Ectoderm
NeuroectodermSB431542, LDN-193189, Y27632 Application Note
Neural progenitorsCHIR99021, SB431542, compound EHuman LIF28132834
Dopaminergic progenitorsThiazovivin, CHIR99021, SB431542, LDN-193189, Purmorphamine, DAPTHuman SHH, Human GDNF, Human BDNF28094017
Neural crest SB431542, LDN-193189, Y27632Human EGF, Human FGF-228762648
Peripheral NeuronsSB431542, LDN-193189, Y27632Human EGF, Human FGF-228762648
Mesoderm
Endothelial cellsY27632, CHIR99021, ForskolinHuman VEGF26214132
CardiomyocytesCHIR99021, IWR-1-endo Application protocol
Hemogenic endotheliumCHIR99021 28302184
Endoderm
Pancreatic beta cellsCHIR99021, LDN-193189, Retinoic Acid, TPPB, Sant-1Human FGF-723897760
Hepatic differentiation Human Activin A, Human BMP-4, Human FGF-2, Human HGF, Human Oncostatin M26586530
Alveolar differentiationCHIR99021, SB431542, Dorsomorphin, IWR-1-endo, LY294002Human Activin A, Human FGF-2, Human FGF-10, Human BMP-4, Human FGF-728802115
In addition to directed differentiation using small molecules and growth factors, mRNA transfection of critical transcription factors can be used to aid differentiation. A number of neuronal and adipocytic mRNAs are available for transfection of PSCs.

8.3 Purification of cells during and after differentiation

At a glance: Kits and reagents for the separation of differentiated cells

Target cell type Starting material Isolation strategy CommentsAutomationProduct
Ectoderm
Neural crestSingle-cell suspension Positive selection of target cellsBased on CD271 expressionYesNeural Crest Stem Cell MicroBeads, human
Dopaminergic progenitorsSingle-cell suspension Two-step positive selection of target cellsBased on IAP/ CD47 expressionYesCD47-PE, human and Anti-PE MicroBeads
Neuronal progenitorsSingle-cell suspension Positive selection of target cellsBased on PSA-NCAM expressionYesAnti-PSA-NCAM MicroBead Kit, human and mouse
Glial progenitorsSingle-cell suspension Positive selection of target cellsBased on A2B5 expressionYesAnti-A2B5 MicroBead Kit, human and mouse
Mesoderm
Hematopoietic progenitorsSingle-cell suspension Positive selection of target cellsBased on CD34 expresYesCD34 MicroBead Kit UltraPure, human
CardiomyocytesSingle-cell suspension Depletion of non-cardiomyocytesDepletion cocktailYesPSC-Derived Cardiomyocyte Isolation Kit, human
EndotheliumSingle-cell suspension Positive selection of target cellsBased on CD144 (VE-Cadherin) expressionYesCD144 (VE-Cadherin) MicroBeads, human
Endoderm
Definitive endodermSingle-cell suspension Positive selection of target cellsBased on CD184 (CXCR4) expressionYesCD184 (CXCR4) MicroBead Kit, human

MACS MicroBeads enable rapid isolation of cells of specific lineage at any time point during differentiation. Optimized kits are available for a number of PSC-derived cells If a particular cell type is not listed, use the MACS Marker Screen, human to screen and analyze 371 human markers and identify suitable ones to isolate the cells of interest. MACS MicroBeads are also available for a wide range of indirect labeling strategies, such as the isolation of dopaminergic progenitors listed above.