Protocol for robust iPSC derivation on Laminin-521 matrix
A robust iPSC derivation protocol form the iPSC core at Karolinksa Institute. Non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.
GMP-ready hepatocyte differentiation protocol
The method presented here by the group of Dr. David Hay, University of Edinburgh, UK, describes a scalable and GMP-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells on human recombinant laminin substrates. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.
A GMP-compliant protocol for production of hESC-derived dopaminergic neurons
Here, Agnete presents a GMP-compliant protocol for neuralising hESCs directly from day 0 of differentiation in a scale suitable for clinical production. This approach not only enables us to get highly pure populations of DA progenitor cells (>90%), but it also increases our final yield of transplantable cells >40 times when compared to previous embryoid body protocols starting with the same number of cells. The protocol is published in Cell Stem Cell.
MESSENGER RNA REPROGRAMMING
- PATH TO CLINICALLY RELEVANT IPSCS
This talk will review the development of mRNA reprogramming from its inception, describe the particular challenges entailed, and address the latest advances which have facilitated implementation of a high-throughput, xeno-free, feeder-free, footprint-free iPSC derivation pipeline based on this exciting technology.
Efficient hepatocyte specification
- improved maturation and functional organization
Here, Kate presents her work on that show that culture of human ES cells on human recombinant laminin-521 and laminin-111 substrates significantly improve hepatocyte differentiation, maturation, function and stabilization of phenotype compared to Matrigel cultured cells. Laminin cultured cells arrange themselves in lobule like structures, express MRP1 and MRP2 and are capable of biliary efflux and show significant effect on hepatocyte P450 enzyme metabolic activity. The GMP-ready hepatocyte differentiation protocol is published in Stem Cell Reports.
Dr. Kate Cameron
PostDoc, University of Edinburgh, UK
Neural stem cells in health and disease
Here, Anna presents how studies of neural stem cells and neurons derived from iPS cells of patients show faithful mimicking of known disease phenotypes in our cellular models of disease, like Alzheimer’s disease, autism, and Down syndrome. She has discovered several novel disease specific cellular phenotypes in their in vitro models, many of them involved in accurate performance of the neural progenitors.
Dr. Anna Falk
Ass. Prof., Karolinska Institutet, Sweden
DNA DAMAGE AND LOW-GRADE MOSAICISM IN HESC CULTURES
Claudia presents data around the frequency and mechanisms behind DNA damage and low-grade mosaicism in hESC cultures. hESC and somatic cell lines frequently acquire segmental copy number variations in the Megabase-scale, forming a non-clonal or low-grade genetic mosaic. The results have been published in Stem Cell Reports showing that hESC grown on laminin-521 show a decreased proneness to acquiring DNA damage during suboptimal culture conditions, such as medium acidification during high culture density.
Dr. Claudia Spits
Ass. Prof., Vrije Universiteit Brussel, Belgium
Efficient pancreatic islet maintenance and expansion
Biologically relevant laminins enable mouse pancreatic islets in vitro culture: expansion, phenotype maintenance and glucose-dependent insulin release. When cultured upon selected combination of islet-specific laminins, pancreatic islets exhibited following effects: (1) robust spreading into flattened adherent clusters with heterocellular organization maintaining local cell contacts, (2) islets, cultured upon the selected combination, maintain functional ability for glucose-dependent insulin release, (3) all the endocrine cell types (alpha, beta, delta, PP-cels) maintain specific markers expression (glucagon, insulin/C-peptide/PDX-1, somatostatin, pancreatic polypeptide) and ability to proliferate.
Clinically compliant hESC-derived RPE cells for advanced macula degeneration
hESC lines derived and cultured on recombinant human laminin 521 under fully chemically-defined and animal substance-free conditions. The differentiated RPE cells exhibit native characteristics including morphology, pigmentation, marker expression, monolayer integrity, polarization and phagocytic activity. The authors are established a large-eyed geographic atrophy model that allowed in vivo imaging of the hESC-RPE and host retina. The protocol for clinical compliant hESC derived RPE cells has been published in Stem Cell Reports.
Dr. Sonya Stenfelt
PostDoc, Karolinska Institute, Sweden
Defined and xeno-free culture of high quality hESC and iPSC
Laminin-521 allow not only self-renewal, but also cloning, derivation, and even clonal derivation of new hES cell lines under defined and xeno-free conditions. Laminin-521 induce fast migration of the cells and laminin isoform specific activation of PI-3K/Akt pathway through interaction with α6β1 integrin. All the hES and human induced pluripotent stem cell lines were confirmed to be genetically stable and pluripotent in in vivo and in vitro assays after extensive culturing on the new substrata. The defined and xeno-free long-term hPSC culture protocol has been published in Nature Communications.
Dr. Sergey Rodin
Senior Researcher, Karolinska Institute, Sweden
Watch as cells grow -
Culture ES and iPS cells on laminin
Stem cell culturing of human ES and iPS cells on recombinant laminin matrices is easy and efficient. Human ES and iPS cells grow as monolayers in a completely defined and xeno-free cell culture environment that enables self-renewal and pluripotency without the use of ROCK inhibitors. Here, we show hES cells plated as single cells on top of recombinant laminin-521, growing from day 0 to day 5 when they are ready to be split again. During the first 1-2 days, a picture was taken every 15 minutes. Days 3-5 a picture was taken every hour.
Tissue specific laminins
Laminins are tissue-specific glycoproteins in the basement membrane, important for cell proliferation, migration and differentiation, and tissue development.
Laminin-521 and laminin-511 are expressed by cells in the inner cell mass of the blastocyst and are therefore optimal as substrates for human pluripotent stem cells. Human recombinant laminin isoforms are developed for defined and xeno-free stem cell culturing and primary cell cultures.
Stem cell culture on laminin-521
Stem cell culturing is easy on human recombinant Laminin-521 (LN521). It facilitates self-renewal of both pluripotent human embryonic stem cells and induced pluripotent stem cells in a chemically defined, feeder-free and animal-protein free cell culture system.
LN521 allows the survival and expansion of human ES and iPS cells after plating from single cell suspension. LN521 based stem cell cultures grow as monolayers on top of the laminin substrate and remain pluripotent without spontaneous differentiation. To culture embryonic stem cells on LN521 enables easy and efficient expansion of your hESCs and iPSCs and provides a biorelevant in vitro stem cell niche.
Derivation and culture of stem cells and primary cells on specific laminins
Professor Karl Tryggvason discusses the use of biologically relevant human recombinant laminins from BioLamina that successfully recreates specific cell niches in the cell culture dish. This enables robust stem cell culture methods that in practical solves all current technical challenges with culturing human ES and iPS cells. It also describes novel data for culturing primary cells such as cardiomyocytes, pancreatic beta cells and endothelial cells.