ES/iPS cells

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.

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).

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.

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.

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.

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.

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).
 

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).

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.

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.

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.

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.
 

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.

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.

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.

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, anti-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.