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

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

mRNA Delivery And the Next Wave of Regenerative Medicine

Published online by Nature Biotechnology, researchers from Ken Chien’s lab at Harvard and other coauthors showed that modified mRNA of VEGF-A injected intramyocardially resulted in the expansion and directed differentiation of endogenous heart progenitors. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients by directing epicardial progenitor cells toward cardiovascular cell types. This publication appears to be the first example of using mRNA as a delivery platform for cell fate-related therapy. AstraZeneca recently invested $240 million on mRNA-related delivery via Moderna, a company with roots within the Harvard stem cell group.

The drastically increased efficacy of using the mRNA platform was accredited to the pulse-like kinetics of mRNA expression profile. It was explained by the fact that native paracrine signals are often transient and precisely regulated in time and space, therefore the pulse-like expression profile of modRNA might be well suited to delivering paracrine-factor signals. Transfected mRNA molecules do not need to penetrate the nuclear membrane, which greatly enhances the efficiency of protein expression on a per transfected molecule over DNA. mRNAs turn over in a much faster pace than plasmid-mediated transgene expression. This is beneficial to many cell fate decisions as exemplified by this recent publication.

Allele Biotech’s reprogramming technologies, licensed by some of the leading stem cell therapy companies, are built around the mRNA platform. We chose mRNA as our core technology to not only change cell fate, but also direct differentiation. We know this platform is the future for cell fate manipulation because we have seen how robustly mRNA expression made the day-and-night difference in gene expression when compared to plasmid DNA (episomal or not), retrovirus, lentivirus, baculo virus, or even transfected proteins. We could convert human fibroblasts into iPSCs, in bulk, in as short as one week with no more effort than changing mRNA complex-containing medium.

Another recent development in iPSC research is in situ reprogramming. Abad et al. generated mice carrying a Tet-inducible cassette of the four cell-reprogramming factors. They then added feed doxycycline to the animals. After several weeks, teratomas appeared in various tissues, indicating that in situ reprogramming had occurred. The iPSCs created this way did not appear to have much advantage over in vitro produced iPSCs other than they are totipotent (helpful if you are studying placenta). Nevertheless, the concept of changing cell fate in situ as dramatically as complete reprogramming is an important leap of faith. As for the next big step, it is easy to see that mRNAs are well suited for in situ reprogramming, as well as transdifferentiation, and more complex gene delivery than the above mentioned VEGF-A alone in heart treatment.

Zangi et al. Nature Biotechnology, http://www.nature.com/nbt/journal/vaop/ncurrent/abs/nbt.2682.html
Abad et al. Nature, http://www.nature.com/nature/journal/vaop/ncurrent/abs/nature12586.html

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Thursday, September 12th, 2013 iPSCs and other stem cells, Viruses and cells No Comments

Allele Biotech Receives $200,000 Grant to Update Its mRNA Reprogramming Commercial Products and Services

On June 10, 2013 Allele received an SBIR award from the National Institute of Drug Abuse (NIDA/NIH) entitled “Revolutionary Technology for Efficient Derivation of Human iPSCs with Messenger RNA”. The goal of the proposed project is to provide to the biomedical research market an advanced reagent kit and services for highly efficient reprogramming of high quality human induced pluripotent stem cells (iPSCs). At the core of this kit is the Allele team’s recent development transcribed messenger RNA (mRNA). Compared to other reprogramming methods, such as lentivirus, Sendai virus, protein, small molecules or any combinations of these reagents, our new generation of the mRNA method often requires less than half the time while sometimes achieving “bulk conversion” efficiency.

While the Allele reprogramming technology was designed for clinical use as the process is feeder-free, xeno-free, chromosome integration-free, as well as without the need for cell splitting, PI, Dr. Jiwu Wang states, “Our purpose of executing the NIH-funded research it to make our method so easy that any researcher can integrate iPSC into his or her projects.” In addition to the extremely high efficiency, mRNA-generated iPSCs should also be more stable because there are no genetic alterations, more uniform among all clones as there is no clonal event, and ultimately suitable for future autologous cell therapy now that creating iPSCs from patient tissue cells should no longer be the rate-limiting steps.

Allele’s business model is to provide cGMP-grade iPSCs to pharmaceutical companies and perform large scale reprogramming by partnering first with university-affiliated hospitals. Great progress has been made in both directions, which has prompted the initiation of a cGMP unit within Allele’s newly acquired building in San Diego.

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Allele Biotechnology Initiates Project On Scaled Manufacturing Of Induced Pluripotent Stem Cells And Differentiation With Chinese Academics

Allele Biotechnology has signed an agreement with Jinan University to develop culturing systems of stem cells and differentiation methods for producing skin tissue cells for wound treatment and stem cell therapy.

San Diego, California (I-Newswire) January 16, 2013 – Allele Biotechnology and Pharmaceuticals, Inc., a San Diego based company with a focus on new technology development, announced today that it has signed an agreement with the Biomedical Institute of Jinan University through a focus group to develop culturing systems of stem cells and differentiation methods for producing skin tissue cells for wound treatment. The joint team will also evaluate using stem cell therapy as potential treatment for arthritis, Lupus, and other autoimmune-related diseases.

Scientists from Allele Biotechnology recently described an important advance in the generation of stem cells capable of producing all the different tissues of the human body. Using messenger RNA molecules and without the need of viral vectors, animal products or feeder cells, this new method can be used to reprogram human fibroblasts into induced pluripotent stem cells (iPSCs). The efficiency is significantly improved over previously reported reprogramming results and the time required to complete reprogramming is slashed in half under optimal conditions.

The Biomedical Institute at Jinan University, a leading comprehensive research university in South China, has focused on translational research in the areas of epidemic diseases and autoimmune diseases. It has broad collaboration with partners and close connections to the biotech industry in China. It was known to have launched (licensed) a number of new biologics in China, and contributed to the understanding and diagnostics of the SARS epidemic in 2003. The institute has also been entitled as the national engineering research center of biopharmaceutics since 2005.

This collaboration will last for at least 2 years, and will go beyond the R&D stage with selected candidates moving into clinical trials, first in China, then in other countries. If the project reaches clinical trials it will be funded jointly by industry and academic partners in the range of $10 million USD.

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Wednesday, January 16th, 2013 iPSCs and other stem cells No Comments