iPSCs and other stem cells

Combining mRNA-Mediated Phenotype Rescue and CRISPR-Created Isogenic Genome

There has been a good volume of publications on using patient-specific iPSCs for disease modeling. Among them, a recent study by Wang et al. published in Nature Medicine is unique because it not only created cardiomyocytes from both Barth Cardiomyopathy patient and wildtype samples for functional analysis, but also combined some of the most exciting new technologies to strengthen the correlation between gene change and disease.

First, functional rescue by mRNA transgene. After mRNA that encodes wild-type cardiolipin aclation enzyme encoding gene tafazzin (TAZ) was transfected into Barth iPSC-CM cells, their defects in mitochondrial functions were corrected. Second, loss of function by genome editing. When CRISPR was used to make genome changes in wildtype cells that mimicked the disease-specific mutation, we recreated the patient’s iPSC-CM phenotype in otherwise wildtype cells. Third, next generation sequencing to confirm genomic changes. And forth, the cardiomyocyte contractibility was assayed on bioengineered chips.

This paper should set an example of how patient iPSCs should be used to create disease models to the fullest extent of usefulness and reliability. We are true believers of the idea that technology development empowers the advancement of science.

“Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies.” Wang, G., McCain, M.L., Yang, L., He, A., Pasqualini, F.S., Agarwal, A., Yuan, H., Jiang, D., Zhang, D., Zangi, L., Geva, J., Roberts, A.E., Ma, Q., Ding, J., Chen, J., Wang, D.Z., Li, K., Wang, J., Wanders, R.J., Kulik, W., Vaz, F.M., Laflamme, M.A., Murry, C.E., Chien, K.R., Kelley, R.I., Church, G.M., Parker, K.K., Pu, W.T. (2014) Nature Medicine, Jun;20(6):616-23. doi: 10.1038/nm.3545. Epub 2014 May 11.

Appearance of iPSCs–Different Reprogramming Stages within the Same Well

Previously scientists at Allele Biotech have reported near uniform conversion of human fibroblasts using our proprietary mRNA mixtures. The first picture below shows a well of cells after 7 days of growing fibroblasts with the new Allele mRNA mix.

This month, by adjusting the mRNA dose while testing Allele’s own reprogramming medium formulation, we observed various stages of cells going through the transition in the same well (see pictures 2 to 5). All stages of reprogramming typically observed over a span of weeks can actually be seen within 1 well of a 6-well plate when we treated human fibroblasts at half the dose of our standard mRNA mix, on day 10, and using Allele Biotech’s new formulation of reprogramming medium.

(1) Warren, Ni, Wang, and Guo 2012 (pdf download)

Previous bulk conversion on Day 7 of reprogramming at full dose mRNA, improved upon our published efficiency (1)

iPSCs forming small colonies from single cells within a 24-hour time frame

Reprogramming en masse: post mesenchymal-to-epithelial (MET) transition cells start to become iPSCs without surrounding fibroblasts (as opposed to the above figure)

Large patches of cells that became iPSCs in what we call bulk-conversion

Large colonies become highly compact, with sharp edges, and composed of mature stem cells of small cell body and tight bundling

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Tuesday, March 11th, 2014 iPSCs and other stem cells 1 Comment

Picture Blog: A Short Path from Human mRNA-iPSCs to Neurons in Record Speed

Traditional differentiation protocols use embryoid body (EB) formation as the first step of lineage restriction to mimic early human embryogenesis, which is then followed by manual selection of neuroepithelial precursors. This procedure is tedious and often inconsistent. We have developed a novel neural differentiation scheme that directs human iPSCs (created with the Allele 6F mRNA reprogramming kit) that progressed, as attached culture, to neural precursor cells (NPCs) in just 4-6 days, half the time it typically takes by other methods. From NPCs it takes about another 5-6 days for neural rosettes to form (see figures below); upon passage, cells in neural rosettes differentiate into neurons in 24 hours.

The neural progenitors at the rosettes stage can be stocked and expanded, before differentiated into different types of neurons. We are working on specifically and efficiently different these neural progenitor cells into dopaminergic, glutamatergic, GABAergic, and other types of sub-types of neurons with Allele’s technologies (Questions? email the Allele Stem Cell Group at iPSatAllelebiotech.com).

Neural rosettes formed efficiently in wells without going through EB.

neural rosettes formed as attached cells in less than 2 weeks

Human iPSC-derived neurons are created in a short regimen developed at Allele Biotech

Neurons appear from precursor cells shortly after the rosette stage

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Wednesday, February 5th, 2014 iPSCs and other stem cells, Open Forum No Comments

Picture Blog: Naive Human Pluripotent Stem Cells Regrown From Allele’s iPSCs

As we blogged a month ago, the Hanna lab recently published a paper in Nature describing that human ESCs or iPSCs, which typically resemble more of mouse EpiSCs (epiblast stem cells) than ground state mouse stem cells, could be converted to naïve pluripotent stem cells if grown in a stem cell medium that includes hLIF, JNKi, and p38i.  The figure here shows that the reported system did perform well when we at Allele Biotech tested growing our banked iPSCs under similar conditions.  The colonies grown in naive stem cell conditions (B) did become dome-shaped when cultured for longer period of time; when transferred back into regular stem cell medium, the once naive-looking iPSCs formed tighter and “cleaner” colonies than typical “primed” human iPSC colonies.

A, Primed human stem cells: mRNA-iPSC line J-1 grown on CellStar-coated surface and in E8 medium. The cells have human iPSC morphology of being compact in size and in "shiny" colonies. B, Naïve human stem cells: J-1 iPSCs shown 2 days after switching to a medium similar to the Naïve Human Stem cell Medium (NHSM). Compared to primed stem cells in A, the naïve stem cells are more flat and transparent, with no spontaneous differentiation on the edges.

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Thursday, December 5th, 2013 iPSCs and other stem cells No Comments

New Allele Biotech Publication on Stem Cells

Feeder-Free Reprogramming of Human Fibroblasts with Messenger RNA
Current Protocols in Stem Cell Biology • November 13, 2013
DOI: 10.1002/9780470151808.sc04a06s27

Authors: Luigi Warren, Jiwu Wang

This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols.

Allele iPSC Service and Technology Licensing Contact: http://www.allelebiotech.com/cell-line-and-culture-services/#ips-line

New Allele Product of the Month: FP-nAb™ products for 100% pull-down

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Wednesday, November 13th, 2013 iPSCs and other stem cells No Comments