A newly-reported breakthrough in technology for generating high-fidelity induced pluripotent stem cells (iPSCs) suggests that these cells will soon be available and safe for use for in people. The implications for regenerative medicine and extending human longevity may be profound.
Background on iPSCs
If you are already familiar with iPSCs and their potentials you may skip this section.
An iPSC is a stem cell created from a normal adult body cell like a skin or blood cell through introduction of transcription factors that hopefully reverts the cell to the epigenomic state of an embryonic stem cell. That epigenomic state involves pluripotency, a condition where that cell can differentiate into any of the hundreds of different body’s cell types. See the March 2009 blog entry Rebooting cells and longevity for my first post on the initial discovery of how to make iPSCs.
If reliable and safe iPSCs that are fully pluripotent could be generated in adequate quantities, the potential for their use in regenerative medicine and for creating significant human longevity could be incredible:
– They might be used to cure genetic diseases. See the blog entries A simple treatment for human genetic diseases and Treating genetic diseases with corrected induced pluripotent stem cells.
– They could be used for all the therapies human embryonic stem cells (hESCs) are being considered for. iPSCs and would be superior to hESCs because they are made from the patient’s own cells and immunologically identical with them, obviating all the possible complications of graft-vs-host-disease which occurs in medical procedures where other people’s cells are used for therapeutic purposes.
– iPSCs might even be used to create extremely longevity through closing the loop in the stem cell supply chain. I have often referred to the blog entries The stem cell supply chain – closing the loop for very long lives, and the follow-up entry Progress in closing the stem cell supply chain loop.
– Finally, iPSCs are free from the religious, moral and political uproars associated with applications using hESCs. The right-to-life people say “Why not use iPSCs instead?” And, speaking as a scientist who believes hESC research should continue, I have to say that they are probably right about this.
The original approach to creating iPSCs, introduction of four cell transcription factors, Oct4, Sox2, Klf4, and c-Myc, had a number of serious problems associated with it including:
– The viral vectors originally used to introduce the transcription factors left traces of their DNA in the resulting cells.
– Other random genetic damage to the cell could be created in the process of cell reversion; there was risk of genomic recombination or insertional mutagenesis.
– The processes of cell reversion were slow and extremely inefficient, converting only a tiny fraction of the cells treated to iPSC status.
– Careful examination of the iPSCs indicated that they were not epigenetically the same as embryonic stem cells and therefore possibly not as pluripotent.
– The problem remained of how to introduce iPSCs into the body so that they differentiate into cell types associated with a particular objective, e.g. to make neural cells to help a Parkinson’s Disease patient, to make heart cells to repair a heart muscle defect, etc. This problem had been identified much earlier with hESCs. If pluripotent cells are simply injected into a body tissue, a teratoma could result which is a hodgepodge tumor of varied tissue types including hair, teeth and bone.
There has been much reported subsequent progress at addressing these issues by new and improved techniques for cell reversion, however none of the approaches overcame all the issues and produced cells sufficiently safe and reliable to be used in humans. Some commercially-available iPSCs, for example, were reported to have short telomeres. See my April 2010 blog entry Induced pluripotent stem cells – second-rate stem cells so far. It looked like iPSCs were good enough for testing drugs but not safe for use in humans. In June 2010 I wrote the blog entry A near-term application for iPSCs – making cell lines for drug testing and in that entry I said “a number of technical challenges must be overcome including: a) obtaining iPSCs that are free of DNA contamination, and that have long telomeres and full hESC pluripotency, b) developing reliable means for assuring differentiation into adult stem cells of various types, and c) developing reliable and safe means for introducing those cells into their respective body niches.
The July 2010 blog entry Induced pluripotent stem cells – developments on the road to big-time utilization reported significant progress in the technology for generating iPSCs and by that time several alternative approaches were known. None, however overcame all of the problems identified above with sufficient reliability to yield iPSCs that could be used in humans, even for experimental purposes. “The search for ways to induce pluripotency without incurring genetic change has thus become the focus of intense research effort. Toward this end, iPSCs have been derived via excisable lentiviral and transposon vectors or through repeated application of transient plasmid, episomal, and adenovirus vectors (Chang et al., 2009,Kaji et al., 2009,Okita et al., 2008,Stadtfeld et al., 2008,Woltjen et al., 2009,Yu et al., 2009). iPSCs have also been derived with two DNA-free methods: serial protein transduction with recombinant proteins incorporating cell-penetrating peptide moieties (Kim et al., 2009,Zhou et al., 2009) and transgene delivery using the Sendai virus, which has a completely RNA-based reproductive cycle (Fusaki et al., 2009)(ref).” None of these approaches completely abrogated the problems identified above, particularly the problem of potential genetic damage or contamination in the resulting iPSC cells. It “become increasingly apparent that all iPSCs are not created equal with respect to epigenetic landscape and developmental plasticity)(ref).”
The new breakthrough development
The required breakthrough is reported in the September 30, 2010 publication Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA. “Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine. — Here we demonstrate that repeated administration of synthetic messenger RNAs incorporating modifications designed to bypass innate antiviral responses can reprogram differentiated human cells to pluripotency with conversion efficiencies and kinetics substantially superior to established viral protocols. Furthermore, this simple, nonmutagenic, and highly controllable technology is applicable to a range of tissue-engineering tasks, exemplified here by RNA-mediated directed differentiation of RNA-iPSCs (RiPSCs) to terminally differentiated myogenic cells.”
Going on, “By using a combination of RNA modifications and a soluble interferon inhibitor to overcome innate antiviral responses, we have developed a technology that enables highly efficient reprogramming of somatic cells to pluripotency and can also be harnessed to direct the differentiation of pluripotent cells toward a desired lineage. Although it is relatively technically complex, the methodology described here offers several key advantages over established reprogramming techniques. By obviating the need to perform experiments under the stringent biological containment required for virus-based approaches, modified RNA technology should make reprogramming accessible to a wider community of researchers. More fundamentally, because our technology is RNA based, it completely eliminates the risk of genomic integration and insertional mutagenesis inherent to all DNA-based methodologies, including those that are ostensibly nonintegrating. Moreover, our approach allows protein stoichiometry to be exquisitely regulated within cultures while avoiding the stochastic variation of expression typical of integrating vectors, as well as the uncontrollable effects of viral silencing(ref).”
In other words, it appears that this new approach that uses modified mRNA to reset cells instead of directly applying transcription factors addresses most of the main issues that have bedeviled human use of iPSCs up to this point. As reported in the popular press “After tinkering with the mRNA molecules in the laboratory to make signals that the cells would not destroy as dangerous invaders, the researchers found that a daily cocktail of their creations were surprisingly fast and efficient at reprogramming the cells. The approach converted the cells in about half the time of previous methods – only about 17 days – with surprising economy – up to 100 times more efficient than the standard approach. — Moreover, detailed tests indicated the cells had not experienced any disturbing changes in their DNA caused by previous methods and were virtually identical to embryonic stem cells. In addition, the researchers went one step further and showed that they could use the approach to then coax the iPS cells they created into a specific type of cell, in this case muscle cells.”
We have to wait for confirming research to be sure there are not other limitations or nasty surprises associated with iPSCs created this new way. And faithful directed differentiation of the iPSCs was actually demonstrated for only one type of muscle cell. But for the moment it looks like there is a real breakthrough. In the June 2010 blog entry I stated that although much research is being devoted to iPSCs, 10-20 years are likely to be required before the stem cell supply chain can truly be closed in humans, the problems being mainly bioengineering in nature.
I still believe the challenges are of a bioengineering nature but there has been so much progress reported since June that I now want to cut my 10-20 year estimate in half. I expect that within 3-5 years we will see experiments with mammals, no-doubt mice to start with, involving use of iPSCs directed to differentiate so as to renew adult stem cells in their niches, the first experiments at closing the loop in the stem cell supply chain. We will also see the first regenerative animal experiments using iPSCs before then, for example the use of iPSCs to regenerate spinal cord tissues and damaged heart valves. And so, in 5-8 years we could see approved human regenerative iPSC therapies. And just possibly, in less than 10 years we will see the first therapies where iPSCs are used to renew adult stem cells in their niches, the initial implementations of the longevity intervention: closing the loop in the stem cell supply chain.