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

What Does It Take to Bring New Nano Antibodies (nAbs) to the Hands of Researchers?

Judging from the hundreds of papers published using camelid VHH antibodies as reagents, there are probably thousands of researchers who have experience with this type of antibodies by now. We like to call the ~15kD camelid VHH antibody nano antibody or nAbTM. Once someone experiences how well a nAb works for co-IP using a fluorescent protein as tag, they often wonder what it takes to bring nAbs to broader use.

The success of a nAb project starts with the antigen presentation. It is critical to build the capability to produce large quantities of recombinant antigen for immunization. At Allele, our scientists also established some unique presentation formats for traditionally difficult targets (e.g. large membrane proteins).

After llama immunization, the next step is screening. With the goal of creating large scale nano antibodies against diverse targets, we have developed multiple high throughput screening methods to cover very large, diverse libraries generated from immunized animals. The technologies will continue to evolve as the scale of nAb generation continues to expand. We have the ability to functionally screen for site-blocking antibodies and antibodies that only recognized natively folded targets, or targets in their naturally occurring presentations.

A nAb isolation project does not end with the obtaining of a cDNA clone. Or, if it does, the nAb is probably not as great as what Allele Biotech has been offering. In our hands, all nAbs go through an engineering step beginning with the generation of a 3D structural model of the isolated clone. We use structure-guided design to alter the protein, allowing us to improve its properties. This includes increasing affinity, solubility, or altering the protein to improve performance for specific applications. We also like to use known structures of traditional monoclonal antibodies to assist camelid VHH antibody engineering against specific targets.

With a finalized clone in hand, the next step is to establish protocols for commercial production. The Allele team spends a tremendous amount of effort aimed solely at high-yield, low-cost recombinant VHH antibody production in a variety of formats, so that the costs for other scientists to take advantage of these great reagents can be kept as low as possible.

Last but not the least, nAb labeling, including conjugating stable soluble VHH antibody to solid supports for immunoprecipitation or to fluorophores for detection, requires additional expertise and tight operation control. However, our vision is to have a modular system for antibody labeling that will enable the end user to select from a variety of fluorophores and other detection tags, which can be instantaneously and irreversibly coupled via simple mixing.

Note added: we work with commercial (diagnostic and clinical) partners from developing nAbs all the way to the market. We have expert scientists available to customers and licensees for consultation and troubleshooting antibody- and imaging-related questions and problems.

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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

Allele Biotech Acquires BioCarta’s Distribution Business

Allele Biotechnology & Pharmaceuticals, Inc. is pleased to announce that as of January 1st, 2014, it has acquired the distribution business of BioCarta. This transaction will strengthen Allele Biotech’s presence in the antibody field, enhancing a broad customer and partnership base to further its plan in clinical diagnostics. Biocarta has been a leader in the field of gene expression for 10 years and has contributed immensely through its world leading effort of charting molecular biology pathways. The gene function maps published by Biocarta have been used and referred to by the NIH through the National Center for Biological Information and National Library of Medicine.

Among its well-regarded distribution business, for the past 11 years BioCarta had been the US and Canada’s exclusive distributor of Immune Function Assay Kits for Flow Cytometric Analysis by Glycotope Biotechnology, GmbH. These kits are clinically approved blood cell diagnostic assay products that are also heavily used for non-clinical blood studies.

Allele Biotech has recently launched a major business plan to create a large number of nano antibodies (nAbsTM). The nAbTM line will be great research tools for immunoprecipitation (co-IP), immunohistochemistry (IHC), Western blotting, co-crystallization, biologics purification. Additionally, nAbsTM willbe suitable for diagnostic assays because the single domain antibodies derived from camelid family animals are sturdy, specific, and low-cost. The inclusion of the BioCarta distribution channels in the antibody and pathway reagent fields will help speed up Allele Biotech’s expansion.

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Thursday, January 16th, 2014 Allele Mail Bag 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