JvH 3 + 3b

RNA virus

Non-pathogenic

Broad tropism

hoofdkenmerken Sendai virus

• Integrating technologies kunnen het genoom veranderen
  → risico op insertional mutagenesis en veranderde celfunctie

• Sommige celtypes zijn moeilijk te reprogrammeren
  → lage efficiëntie, zeker bij beperkte patiëntsamples

• Sommige methodes zijn technisch minder praktisch
  → bijvoorbeeld meerdere transfecties nodig

Principe: een goede reprogrammingmethode moet efficiënt, veilig en praktisch uitvoerbaar zijn, zonder permanente genetische schade aan de cellen.

reprogramming challenges

Sendai Reprogramming Kit

een eerste kit dat de hoofdchallenges van reprogramming aanpakt

Reprogram somatic cells into iPSCs using transcription factors.

What is the main purpose of the CytoTune-iPS Sendai kit?

Non-integrating system, so no insertion into the genome.

Why is Sendai reprogramming often preferred over integrating viral methods?

GMP version is for clinical/translational use, research version for standard lab use.

What is the difference between GMP and normal research versions of the Sendai reprogramming kit?

Cells should stay pluripotent in culture and only differentiate when intentionally induced.

Why is spontaneous differentiation a problem during pluripotent stem cell culture?

Non-optimal medium or culture conditions.

What can cause unwanted differentiation of pluripotent stem cells in culture?

They survive better and experience less stress than single isolated cells.

Why are pluripotent stem cells often passaged as clumps/aggregates instead of single cells?

Methods like electroporation often require single cells, but single-cell survival is poor.

Why is engineering/editing pluripotent stem cells challenging?

Culture of pluripotent stem cells.

What is E8 medium used for?

Chemically defined composition (components are exactly known).

What is an important advantage of E8 medium?

FGF in the medium is not very stable.

Why does E8 medium often need daily refreshing?

The medium itself is costly and must often be refreshed daily.

Why is pluripotent stem cell culture with E8 considered expensive?

Version of E8 with more stable FGF, allowing less frequent feeding.

What is the main idea behind E8-Flex?

Optimized culture medium for human pluripotent stem cells (PSCs).

What is StemFlex medium mainly designed for?

StemFlex medium

• Geoptimaliseerd voor hPSC/iPSC-cultuur
  → robuuster dan standaard E8-type media

• Ondersteunt moeilijke workflows beter:

  • single-cell passaging

  • gene editing

  • reprogramming

  • recovery na stress/thawing

• Bevat stabielere/protectieve componenten
  → cellen overleven beter en differentiëren minder spontaan

• Nadeel: duurder en deels proprietary
  → je weet niet altijd exact wat erin zit

Principe: StemFlex is een “top” medium omdat het hPSCs stabieler en stressbestendiger houdt, vooral bij technisch moeilijke stappen zoals single-cell passaging en gene editing.

Why is StemFlex often considered a “top” medium?

Single-cell passaging, gene editing, and reprogramming.

For which PSC workflows is StemFlex specifically promoted?

It performs well across many PSC applications.

Why do some labs use StemFlex for everything?

Very expensive.

What is the main disadvantage of StemFlex?

For attachment and growth on the culture plastic.

Why do pluripotent stem cells need a matrix coating?

They are derived from mouse tumor matrix and are variable between batches.

Why are Matrigel/Geltrex mainly used for lab research and not patients?

More defined and xeno-free culture conditions, better suited for clinical use.

What is an advantage of recombinant matrices (eg vitronectin/laminin)?

Clump passaging

• Pluripotente stamcellen worden niet volledig losgemaakt tot single cells

• Kolonies worden in kleine stukjes/clumps verdeeld

• Die clumps worden opnieuw uitgezaaid op een nieuwe culture plate

• Dit geeft minder stress en betere overleving
  → hPSCs overleven slecht als losse single cells

Principe: klassiek worden pluripotente stamcellen gepasseerd als kleine koloniegroepjes, omdat ze beter overleven wanneer ze cel-celcontact behouden.

What is the classical passaging method for pluripotent stem cells?

Single pluripotent stem cells survive less well and need supportive additives.

Why is single-cell passaging more challenging than clump passaging?

Normal morphology, pluripotency markers, and normal karyotype.

Which properties should remain stable during long-term PSC culture?

Contains a ROCK inhibitor that improves survival of stem cells.

What is the role of RevitaCell in PSC culture?

Single-cell dissociation can trigger stress/apoptosis.

Why is a ROCK inhibitor often used after single-cell dissociation?

• Reguleert het actin cytoskeleton
  → bepaalt celvorm en spanning

• Stuurt cell contraction
  → cellen kunnen samentrekken

• Beïnvloedt cell survival
  → vooral belangrijk na single-cell dissociation

• Bij hPSCs kan te veel ROCK-activiteit stress/apoptose geven na passaging

Principe: ROCK-regulatie bepaalt hoe cellen omgaan met cytoskeletstress; daarom verbeteren ROCK inhibitors de overleving van hPSCs na single-cell passaging of thawing.

What does the ROCK pathway regulate?

Two ways of working: from a healthy cell, adding the variant, or the other direction (having both is the best option, where you need genome editing for both)

hoe werkt genome editing in stem cells?

Improves survival during single-cell cloning and after genome editing.

What is CloneR used for in pluripotent stem cell culture?

Feeder vs feeder-free culture

attachment substrate

passaging method

dissociation reagent

large-scale culture conditions.

What are the main culture variables pluripotent stem cells are sensitive to?

Culture without support feeder cells, using defined medium and matrix instead.

What is feeder-free culture?

Some believe feeders better support pluripotency or cell quality.

Why do some groups still use feeder cells?

Clump passaging transfers colonies in groups, single-cell passaging dissociates cells individually.

What is the difference between clump passaging and single-cell passaging?

Higher survival and less stress for pluripotent stem cells.

What is an advantage of clump passaging?

To produce large numbers of cells at larger scale.

Why are bioreactors used in stem cell culture?

The exact components and composition are known.

What does it mean that E8 medium is chemically defined?

It contains 8 key components for hPSC maintenance.

Why is E8 called E8?

Maintenance/culture of human pluripotent stem cells.

What is E8 medium used for?

By STEMCELL: A similar defined medium as the E8 from ThermoFisher for human pluripotent stem cell culture.

What is mTeSR1?