Stem cell culture
Human pluripotent cells grow as colonies on a defined coating (Matrigel, laminin or vitronectin) in a defined medium such as E8 or mTeSR, fed daily and passaged as clumps with EDTA before the colonies touch.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Feeder-free culture
Human pluripotent cells grow as colonies on a defined coating (Matrigel, laminin or vitronectin) in a defined medium such as E8 or mTeSR, fed daily and passaged as clumps with EDTA before the colonies touch.
How it differs from the standard method
- Replaces mouse embryonic fibroblast feeders and serum-based medium with defined components.
- Coatings like Matrigel must be kept cold until plated.
- Single-cell passaging needs a ROCK inhibitor and tends to select culture-adapted variants faster.
1Cells that can become something else
A stem cell divides to make more of itself and can also give rise to specialised cells. Pluripotent stem cells, either embryonic stem cells from the inner cell mass of a blastocyst or induced pluripotent stem cells (iPSCs) reprogrammed from adult cells, can in principle make any cell type of the body.
Reprogramming
OCT4, SOX2, KLF4 and c-MYC switch an adult cell's identity back to pluripotency over three to four weeks.
Feeder-free culture
A defined medium and a coating replace the mouse fibroblast feeder layer of early stem cell work.
Directed differentiation
Small molecules that switch developmental pathways on and off in sequence walk the cells through embryonic stages.
2Making iPSCs and directing their fate
Reprogramming delivers the four factors, today usually as non-integrating Sendai virus, episomal plasmids or mRNA, into fibroblasts or blood cells. Only a small fraction of cells (often 0.01–1%) complete the process; many colonies stop part-way and look convincing while lacking markers such as TRA-1-60.
3How it goes wrong
- Colonies left to merge, or medium left unchanged over a weekend: flat, granular differentiated areas spread from the edges and centres.
- Single-cell passaging without a ROCK inhibitor: most cells die within hours.
- Differentiated cells carried from passage to passage: they induce more differentiation.
- Long or harsh culture: variants such as gains of 20q11.21 take over without changing how the cells look.
- Reprogramming low-passage cells too late or high-passage cells at all: few colonies.
- Picking by eye too early: partly reprogrammed clones that fail later.
- Differentiation at the wrong density or CHIR dose, or in insulin-containing medium: few cells of the target lineage.
- A marker gate without a negative control: a number that is not a measurement.
Key terms
Practise these as flashcards →The ability to give rise to cells of all three germ layers; maintained by OCT4, SOX2 and NANOG and shown by markers such as TRA-1-60 and SSEA-4.
Induced pluripotent stem cell: a somatic cell reprogrammed to pluripotency by expressing defined transcription factors.
A drug such as Y-27632 that blocks Rho-associated kinase and prevents the apoptosis of dissociated human pluripotent cells.
An RNA virus vector that expresses reprogramming factors in the cytoplasm without integrating, and is gradually lost as cells divide.
Driving pluripotent cells towards one fate with a timed series of growth factors or small molecules that mimic development.
Selection of genetic variants (for example 20q11.21 gain, trisomy 12 or 17) that grow better in culture; detected only by karyotyping or copy-number assays.
A floating aggregate of pluripotent cells that differentiates spontaneously into cells of all three germ layers; used to test pluripotency.
When to use it
- When the cell type you need (human cardiomyocytes, neurons, hepatocytes) cannot be obtained from donors in useful numbers.
- When you want a patient's own genetic background, for disease modelling or to compare edited and isogenic control lines.
- When you need an unlimited, renewable source of a defined human cell population for screening.
Compared with alternatives
Primary cells are mature and real but finite; iPSC-derived cells are renewable but often less mature than their adult counterparts.
Many organoids are grown from pluripotent cells; once differentiated in 3D they give tissue architecture that a monolayer lacks.
Routine passaging of immortal lines is far more forgiving; pluripotent cells need daily feeding, clump passaging and removal of differentiation.
Flow cytometry with proper negative controls is how pluripotency and differentiation efficiency are quantified.
Reprogramming factors are delivered by viral or non-viral vectors; integrating lentiviral delivery is avoided for lines meant to be transgene-free.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Human pluripotent cells grow as colonies on a defined coating (Matrigel, laminin or vitronectin) in a defined medium such as E8 or mTeSR, fed daily and passaged as clumps with EDTA before the colonies touch.
- Replaces mouse embryonic fibroblast feeders and serum-based medium with defined components.
- Coatings like Matrigel must be kept cold until plated.
- Single-cell passaging needs a ROCK inhibitor and tends to select culture-adapted variants faster.