Induced Pluripotent Stem Cells (iPSC) for Variant Biology Discovery – Dramatic Increases in Efficiency Are Starting to Occur!

guest co-author: Trisha Brock

The use of iPSC cells is quickly gaining momentum as tool of personalized medicine

Whether you call them stem-cell-like cells with unique expression signatures (Chin 2009), or if you stick with the seminal publication and call them induced Pluripotent Stem Cells (Takahashi 2007), iPSC technology is becoming an impressive system for helping understand genome-encoded disease biology.  We can expect iPSCs to have major impact in regenerative medicine, disease modeling, and drug development.

iPSC technology is a relatively new technique for variant profiling.  

Since inception with demonstration in mice (Takahashi 2006), progress has occurred rapidly. Original systems were developed using retroviral integration tools, which have high risk of chromosomal instability and tumorigenesis. Then, in the last 10 years, significant progress has been made to improve the process by making the technique non-integrative and more efficient (Omole and Fakoya 2018).  Genomic integration methods using viruses have traditionally yielded the highest efficiency, but non-integrative methods are quickly catching up. The original methods used a standard set of gene expression modifiers: Oct3/4, Sox2, Klf4, and c-Myc – typically referred to as the “OSKM cocktail.” These are encoded in viruses and plasmids that are transfected into the cell and integrate into the genome. This one time procedure contrast with non-integrating methods, which require repeat transfections to boost cells towards pluripotency (Warren 2010).  Once induced into the pluripotent state, the cell can be modified (CRISPR-based gene editing or similar methods) to establish control lines with and without variant in question. Finally cells are differentiated into desired tissue by exposure to appropriate tissue-specification factors. With this capacity to analyze patient derived tissue, you can feel fairly certain the biological differences seen between control and variant will translate well to the patient’s specific biology.

Adapted from El Hokayem 2016

Speed to variant biology data requires 2 to 5 months

Relative to rodent animal models, the system is fast.  Most human cell cultures can be enticed to achieve the proper pluripotent expression profile after about 4 weeks of growth and then there is another 2 months or more to induce desired tissue type.  For instance, in a popular method, DeRosa et al. used blood-derived Sendi-virus-transformed iPSCs to derived cortical neurons and test them for biological consequence with various functional assays (DeRosa 2018).   Biomarkers for transformation into iPSCs were cell-stain-cofirmed with antibodies for NANOG, Oct 3/4, and SOX2. Next, cells were differentiated to desired cortical neuron tissue type by exposure to relevant co-factors and monitored for sequential biomarkers production (Nestin to DCX to CAMK2A to TBR1) and finally, at 90 days, the last set of biomarkers was used (MAP2 and SYNAPSIN1).  The result, the creation of iPSCs and their differentiation into appropriate tissue types takes about 3 months or more, depending on the desired cell type needed (McKinney 2017 and DeRosa 2018).

Low efficiency problem of easily sourced iPSCs is showing signs of dramatic improvement

Low efficiency in creation of iPSCs has been the main drawback preventing routine use as a clinical diagnostic.  Efficiency is measured as number of iPSC cells obtained after dividing by the number of input cells prior to starting transformation. Efficiencies vary widely with multiple reports range from 0.1% to 0.001% (Malik and Rao 2013). The measured efficiency is heavily influenced by choice of starting material. Fibroblast show some of the highest efficiencies but typically require a biopsy plug from the skin to enable isolation of sufficient amounts of starting cells. Yet, researchers are hard at task working on conditions that improve efficiency and find easier to source material.  Two years back, a report published conversion of fibroblasts had improved to an efficiency near 3% (Pomeroy 2016). Isolation of iPSC is more convenient to the patient source of blood sources (PBMCs) but efficiency remain low at 0.15–0.32% (Zhou 2015). Generation of iPSCs from PBMCs is problematic because they are non adherent. For human cells, the lack of adherence is especially problematic because it leads to high activation of cell death through apoptosis. Researcher are finding the use of adhesion promoting matrices (Geltrex and rhLaminin-521) and ROCK inhibitors of apoptosis can greatly improve the efficiency process for making iPSCs from blood sources (Ye 2018). Additional recent developments are highly encouraging. For a nearly nonexistent invasiveness to the patient, researchers have demonstrated iPSC can be sourced from patient urine (Gaignerie 2018). Further, in a very impressive efficiency feat, a meticulous study of fibroblast cell conversion was performed by sampling a wide variety of optimizing conditions (Kogut 2018).  Using mRNA encoded transformation factors with a select set of microRNA inhibitors of transcription, the Kogut team demonstrated an amazing 80% efficiency in fibroblast conversion to iPSC. Additionally, they found they could isolate single cells and get conversion to iPSC in 90% of the isolates. Intriguingly, in an almost counter-intuitive finding, efficiency of fibroblast conversion started to drop dramatically when more than 1000 source cells were present at the starting conditions. Finally, their speed to conversion of source to iPSCs was only 15 days. In conclusion, it appears iPCS methodologies are breaking through the efficiency barrier and soon the use of patent-derived iPSCs will become part of routine clinical diagnostic procedures.

Transplantation of iPSCs as therapeutics is challenged by tumorigenics issues.

Three main applications of iPSC are its promise for regenerative medicine, disease modeling, and drug development, yet transplantation for regenerative medicine has issues of tumorigenesis to overcome (Focosi 2018).  The original work by Takahashi was plagued by significant tumorigenicity, because the method uses genomic-integrating viral vectors with transformation factors known for their tumorigenesis potential – mouse iPSC derived from the method would result in tumors at 20% of the time when the cells were reintroduced into mice (Omole and Fakoya 2018). Switching to a transformation cocktail that avoids the use of cMyc (OSKM to OSNL) reduced tumor formation rates.  Yet the alternative transformation factors do not eliminate tumorigenesis, possibly due to genomic integration causing insertional mutagenesis. To reduce tumorigenicity even further, researchers have been switching to non-integrative methods. Yet even then, a low level of tumorigenic capacity remains – apparently inherent to iPSC cells “totally potent” status, where they can become many different types of tissue, including cancerous ones. As a result, when differentiating iPSC into tissue for reintroduction into the patient as treatment, the FDA remains concerned about the tumorigenic potential of any cells that remain in the iPSC state. Current thoughts are that a 10-fold passage of a differentiated cell population may effectively eliminate the tumorigenic risk, yet this need for high passage number has tempered the enthusiasm for use of iPSCs for regenerative medicine applications.

Polygenic Consequence – iPSCs from patient tissues has unique advantage that the multiple “Risk Factors” variations of the patient background are retained.

One big advantage to using patient-derived tissue is the genetic background of the test system is exactly as occurs in the patient. For instance, DeRosa and team studied Autism-related variants using patient-derived iPSCs differentiated into neuronal tissues (DeRosa 2018). They looked at 6 patients with variants in target genes suspected of involvement in Autism Spectrum Disorder.  Genomic data was provided on 5 of the patient conditions (see Table below). All suspect variants were observed as heterozygotes. As a result, clear pathogenicity of any variant is lacking, when referenced against existing databases sources. Nevertheless, they saw clear phenotypic consequence of all 6 cell lines examined in their studies (RNA-seq, multi-electrode array recordings, spontaneous calcium transients and scratch recovery assays).

In what might be a highly recommended next step, the De Rossa authors could use CRISPR on their iPSC lines to make isogenic controls.  For instance, on cell line 377110, the VPS123B variation is a prime candidate for using CRISPR-Cas9 gene editing techniques to reverse the rs28940272 locus back to Asparagine.  If this isogenic control behaved as wildtype in all the phenotyping assays deployed, then the authors would have generated a definitive demonstration that the Asn2993Ser variant in VPS13B is pathogenic.

Modeling functional defects in autism variants could be done in C elegans (see Table below).  5 of the 8 genes in the DeRosa work have homology that exceeds 42%. The PRICKLE1 gene has sufficient similarity. Either its Val57Phe or Glu185Ter variations could be installed into a PRICKLE1-humanized animal model.  The Glu185Ter would very likely model a loss-of-function allele effect, but Val57Phe is a missense variant that could go either way. The change of valine to a phenylalanine may disrupt by either gain-of-function, or it may cause a loss-of-function leading to quite different phenotype in functional assays. Only testing directly in an a model system will be the way to get the needed definition of mechanism of action.

Modeling epilepsy with iPSCs requires 135 day preparation protocol

iPSC cell technology will likely become a widespread tool of personalized medicine and it holds significant promise for neuronal regeneration (Wu 2018).  Although it suffers from challenges of asynchronicity, tumorigenicity, timeliness, and low efficiency (Vitrac 2018, Omole and Fakoya 2018), much progress has been made, as attested with this article’s focus on the DeRosa paper.

To list aspects of concern and advantage with iPSC tech, we have:

  • Tumorigenicity and Immunogenicity.  This is especially an important concern in regards to regenerative medicine uses of iPSC.  Perhaps the most promising approaches involve the use if various RNA molecules to provide the requisite reprogramming factors at minimal potential for tumorigenicity and immunogenicity.
  • Clinical Diagnostic.  Sendai virus tech seems to be one of the most promising methods.  Stability reprogramming factors is high, so repetitive redosing is not as problematic.  Also sourcing from easy to acquire primary culture (blood, urine, and saliva) is promising for minimizing the procedure’s invasiveness to the patient.
  • Isogenic Control.  CRISPR-Cas9 tech can be used to return a suspect variant back to wild type.  Functional studies on the variant strain and its isogenic control will determine if the variant is pathogenic or benign.

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