By Vince Giuliano
The last few months have seen the continuation of an extraordinary pace of research related to induced pluripotent stem cells (iPSCs), a technology stream that has been widely heralded as key for organ regeneration, for regenerative medicine, and that, as I have pointed out, has the potential to enable very long lives. This blog entry relates to selected publications that have emerged in just the last few months.
Background on iPSCs
iPSCs are stem cells created from normal body cells like skin cells by resetting their epigenomic state to close to that of an embryonic stem cell using transcription factor proteins, typically Oct4, Sox2, Klf4, and c-Myc. iPSCs were first derived from mouse cells in 2006 and from human cells in 2007. iPSCs were at first beset by numerous problems. Efficiency of reprogramming was very low. The cells appeared not to be fully pluripotent (capable like hESCs to differentiate into all somatic cell types). And they were genetically contaminated making them potentially oncogenic. These factors rendered iPSCs unsuitable for human clinical purposes and even ruled out research studies that administered iPSCs to humans. Because of the potential applications for iPSCs in regenerative medicine, however, researchers begin to address these and other problems, slowly vanquishing them.
“In February 2008, in ground-breaking findings published in the journal Cell, scientists announced the discovery of a technique that could remove oncogenes after the induction of pluripotency, thereby increasing the potential use of iPS cells in human diseases. In April 2009, it was demonstrated that generation of iPS cells is possible without any genetic alteration of the adult cell: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency. The acronym given for those iPSCs is piPSCs (protein-induced pluripotent stem cells)(ref).”
A search in this blog using the term iPSCs will reveal dozens of blog postings relating to them starting in March 2009. In September 2009 I first outlined how very long lives might be enabled by using induced pluripotent stem cells (iPSCs) to do what I called “closing the loop in the stem cell supply chain.” My latest Update on induced pluripotent stem cells was published June 10, 2011. This present blog entry focuses on developments that were published in only the last four months or that I have not reported on previously.
Methods are being discovered to improve the efficiency of production of iPSCs and the quality and purity of the resulting cells.
The traditional methods for reverting somatic cells to iPSC status are notoriously inefficient. “– the throughput of successfully reprogrammed cells has been incredibly low. For example, the rate at which somatic cells were reprogrammed into iPS cells in the Yamanaka mouse study was .01-.1%. The low efficiency rate may reflect the need for precise timing, balance, and absolute levels of expression of the reprogramming genes. It may also suggest a need for rare genetic and/or epigenetic changes in the original somatic cell population or in the prolonged culture(ref).” Further, as I have previously reported, iPSCs have tended to suffer variable pluripotency, genomic instability, unwanted genetic and epigenetic modifications acquired during reprogramming, and have tended to be potentially oncogenic.
The September 2011 publication Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1 reports dramatic improvement in reprogramming efficiency through the use of two additional reprogramming transcription factors. “Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. Here we report that enhancing RA signaling by expressing RA receptors (RARs) or by RA agonists profoundly promoted reprogramming, but inhibiting it using a RAR-α dominant-negative form completely blocked it. Coexpressing Rarg (RAR-γ) and Lrh-1 (liver receptor homologue 1; Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells to ground-state iPSCs requires only 4 d induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous factor-independent iPSCs, which resembled ground-state mouse ES cells in growth properties, gene expression, and signaling dependency. Our findings demonstrate that signaling through RARs has critical roles in molecular reprogramming and that the synergistic interaction between Rarg and Lrh1 directs reprogramming toward ground-state pluripotency.”
An October 7 press release from the Wellcome Trust Sanger Institute relating to the same research, Seeking superior stem cells — 100-fold increase in efficiency in reprogramming human cells to induced stem cells, reports: “Their process increases the efficiency of cell reprogramming by one hundred-fold and generates cells of a higher quality at a faster rate. — Until now cells have been reprogrammed using four specific regulatory proteins. By adding two further regulatory factors, Liu and co-workers brought about a dramatic improvement in the efficiency of reprogramming and the robustness of stem cell development. The new streamlined process produces cells that can grow more easily. — “The reprogrammed cells developed by our team have proved to have the same capabilities as mouse stem cells,” states Pentao Liu, senior author from the Sanger Institute. — The team’s technology produced reprogrammed cells after just four days, compared to the seven days required for the four-protein approach. Key indicators of successfully reprogrammed cells, Oct4 and Rex-1 genes, were seen to be switched on much faster in a much higher number of cells, demonstrating increased efficiency in reprogramming.”
Other research teams have also been discovering means for improving both the efficiency of reprogramming to iPSCs and the purity and quality of the ensuing iPSC cells. The August 2011 publication Radical Acceleration of Nuclear Reprogramming by Chromatin Remodeling with the Transactivation Domain of MyoD reports: “Induced pluripotent stem cells (iPSCs) can be created by reprogramming differentiated cells through introduction of defined genes, most commonly Oct4, Sox2, Klf4, and c-Myc (OSKM). However, this process is slow and extremely inefficient. Here, we demonstrate radical acceleration of iPSC creation with a fusion gene between Oct4 and the powerful transactivation domain (TAD) of MyoD (M3O). Transduction of M3O as well as Sox2, Klf4, and c-Myc into fibroblasts effectively remodeled patterns of DNA methylation, chromatin accessibility, histone modifications, and protein binding at pluripotency genes, raising the efficiency of making mouse and human iPSCs more than 50-fold in comparison to OSKM. These results identified that one of the most critical barriers to iPSC creation is poor chromatin accessibility and protein recruitment to pluripotency genes. The MyoD TAD has a capability of overcoming this problem. Our approach of fusing TADs to unrelated transcription factors has far-reaching implications as a powerful tool for transcriptional reprogramming beyond application to iPSC technology.”
A July 2011 Science Daily article on this same research involving the TAD of MyoD reports: “The U of M researchers found that by fusing two proteins — a master stem cell regulator (Oct4) and a fragment of a muscle cell inducer (MyoD) — they succeeded in “powering up” the stem cell regulator, which can dramatically improve the efficiency and purity of reprogrammed iPS cells. — “Our team discovered that by fusing a fragment of the powerful protein MyoD to Oct4 we could create a ‘super gene’ which would improve the iPS reprogramming process,” said senior author Dr. Nobuaki Kikyo, Stem Cell Institute researcher and University of Minnesota Medical School associate professor. “The result is what we termed M3O, or ‘super Oct4’ — a gene that improves the creation of iPS cells in a number of ways. In the process we shed new light on the mechanism of making iPS cells.” — The efficiency of making mouse and human iPS cells was increased over 50-fold compared with the standard OSKM combination. Increasing purity. The purity of the iPS cells was much higher with the M3O-SKM gene introduction (98% of the colonies) compared with OSKM (5%). Facilitating the reprogramming. iPS cell colonies appeared in around five days with M3O-SKM, in contrast to around two weeks with OSKM. Decreasing the potential for tumor formation. M3O achieved high efficiency of making iPS cells without c-Myc, an oncogene that can potentially lead to tumor formation. — In addition, human iPS cells usually require co-culture with feeder cells typically prepared from mouse cells, obviously creating a problem when the cells are destined for human transplantation. The M3O model did not require such feeder cells, greatly simplifying the process.”
Factors are being discovered that determine the pluripotency of both human embryonic stem cells and iPSCs.
The September 2011 editorial comment in Cell,Splicing up Pluripotency,relates “In this issue of Cell, Gabut and colleagues (2011) identify a new splice variant of FOXP1 that directly regulates the expression of pluripotency genes. It endows human embryonic stem cells with their pluripotent nature and is required for the reprogramming of somatic cells to induced pluripotent stem cells.” The publication itself is An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming: “Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs.”
Whole-genome sequencing suggests that genomic instability and excessive mutations are not necessarily associated with iPSCs
A very recent (October 2011) publication Genome Sequencing of Mouse Induced Pluripotent Stem Cells Reveals Retroelement Stability and Infrequent DNA Rearrangement during Reprogramming.reports: “The biomedical utility of induced pluripotent stem cells (iPSCs) will be diminished if most iPSC lines harbor deleterious genetic mutations. Recent microarray studies have shown that human iPSCs carry elevated levels of DNA copy number variation compared with those in embryonic stem cells, suggesting that these and other classes of genomic structural variation (SV), including inversions, smaller duplications and deletions, complex rearrangements, and retroelement transpositions, may frequently arise as a consequence of reprogramming.Here we employ whole-genome paired-end DNA sequencing and sensitive mapping algorithms to identify all classes of SV in three fully pluripotent mouse iPSC lines. Despite the improved scope and resolution of this study, we find few spontaneous mutations per line (one or two) and no evidence for endogenous retroelement transposition. These results show that genome stability can persist throughout reprogramming, and argue that it is possible to generate iPSCs lacking gene-disrupting mutations using current reprogramming methods.”
Creation of disease-specific iPSCs offers new opportunities for modeling diseases and drug development.
The May 2011 publication iPSC technology: platform for drug discovery. Pointreports “Disease-specific pluripotent stem cells are a source of affected cell types for drug discovery. The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) provides a platform for generating cells from patients with different disease severities and drug metabolism responses. Although challenges remain for efficient differentiation and high-throughput scale-up, we propose that iPSCs have distinct advantages over embryonic stem cells (ESCs) for evaluating drugs by performing clinical trials on a dish.
The May 2011 publication The use of induced pluripotent stem cells in drug developmentis another review publication on the same topic. “This review focuses on the recent advancements in iPSC technology including disease modeling and control setting in its analytical paradigm. We describe how iPSC technology is integrated into existing paradigms of drug development and discuss the potential of iPSC technology in personalized medicine.”
The September 2011 publicationHematopoietic cells as sources for patient-specific iPSCs and disease modelingreports: “In addition to being an attractive source for cell replacement therapy, human induced pluripotent stem cells (iPSCs) also have great potential for disease modeling and drug development. During the recent several years, cell reprogramming technologies have evolved to generate virus-free and integration-free human iPSCs from easily accessible sources such as patient skin fibroblasts and peripheral blood samples. Hematopoietic cells from umbilical cord blood banks and Epstein Barr virus (EBV) immortalized B lymphocyte repositories represent alternative sources for human genetic materials of diverse backgrounds. Ability to reprogram these banked blood cells to pluripotency and differentiate them into a variety of specialized and functional cell types provides valuable tools for studying underlying mechanisms of a broad range of diseases including rare inherited disorders. Here we describe the recent advances in generating disease specific human iPSCs from these different types of hematopoietic cells and their potential applications in disease modeling and regenerative medicine.”
There is significant research directed ultimately at creating clinical applications of iPSCs for a number of disease conditions.
One of these applications is repair of myocardial damage due to heart disease. The October 2011 publication Human pluripotentstem cell-based approaches for myocardial repair: from the electrophysiological perspectivereports “Heart diseases are a leading cause of mortality worldwide. Terminally differentiated adult cardiomyocytes (CMs) lack the innate ability to regenerate. Their malfunction or significant loss can lead to conditions from cardiac arrhythmias to heart failure. For myocardial repair, cell- and gene-based therapies offer promising alternatives to donor organ transplantation. Human embryonic stem cells (hESCs) can self-renew while maintaining their pluripotency. Direct reprogramming of adult somatic cells to become pluripotent hES-like cells (also known as induced pluripotent stem cells or iPSCs) has been achieved. Both hESCs and iPSCs have been successfully differentiated into genuine human CMs. In this review, we describe our current knowledge of the structure-function properties of hESC/iPSC-CMs, with an emphasis on their electrophysiology and Ca(2+) handling, along with the hurdles faced and potential solutions for translating into clinical and other applications (e.g., disease modeling, cardiotoxicity and drug screening).”
The October 2011 publication Transplanted Induced Pluripotent Stem Cells Improve Cardiac Function and Induce Neovascularization in the Infarcted Hearts of db/db Mice reports: “Recently, we proclaimed that induced pluripotent stem (iPS) cells generated from H9c2 cells, following transplantation into infarcted nondiabetic mice, can inhibit apoptosis and differentiate into cardiac myocytes. iPS cells can be an ideal candidate to expand regenerative medicine to the clinic. Therefore, examining the wide range of their potential to differentiate into neovascular cell types remains a major interest. We hypothesized that transplanted iPS cells in the infarcted diabtetic db/db and nondiabetic mice can differentiate into vascular smooth muscle (VSM) and endothelial cells (ECs) as well as activate endogenous c-kit progenitor cells to enhance neovascularization along with improved cardiac function. We transplanted intramyocardially 50,000 iPS cells in the peri-infarct zone of infarcted db/db and C57BL/6 mice and hearts were examined at D14 post-MI. Cardiac function was examined using echocardiography. Our data implies that there was a significant (p < 0.001) increase in VSM and ECs in the infarcted heart following iPS cell transplantation compared with MI and sham groups in both db/db and C57BL/6 animals. Furthermore, the MI+iPS cell transplanted group also displayed a significant (p < 0.001) increase in c-kit(+ve) activated VSM and ECs confirmed with combined stainings of c-kit and cell specific markers, compared with respective controls. Next, our histology data in the MI+iPS cell group also establishes a significant (p < 0.05) increase in coronary artery vessels compared with MI, suggesting neovascularization. Furthermore, our data demonstrates significant improved cardiac function following iPS cell transplantation compared with MI. Overall increased neovascularization in the infarcted db/db and C57BL/6 mice is associated with improved cardiac function following iPS cell transplantation.”
The above-mentioned publication relates to repair of vascular damage and restoration of cardiac damage due to diabetes, albeit in a mouse model. Another issue related to treatment of diabetes is restoration of normal insulin functioniong. This issue is addressed in the October 2011 review publication Insulin-producing Surrogate β-cells From Embryonic Stem Cells: Are We There Yet? “Embryonic stemcells (ESCs) harbor the potential to generate every cell type of the body by differentiation. The use of hESCs holds great promise for potential cell replacement therapies for degenerative diseases including diabetes mellitus. The recently discovered induced pluripotent stem cells (iPSCs) exhibit immense potential for regenerative medicine as they allow the generation of autologous cells tailored to the patients’ immune system. Research for insulin-producing surrogate cells from ESCs has yielded highly controversial results, because many steps and factors in the differentiation process are currently still unknown. Thus, there is no consensus on common standard protocols. The protocols presently used established the differentiation from pluripotentcells toward pancreatic progenitor cells. However, none of the differentiation protocols reported to date have generated by exclusive in vitro differentiation sufficient numbers of insulin-producing cells meeting all essential criteria of a β-cell. The cells often lack the crucial function of regulated insulin secretion upon glucose stimulation. This review focuses on past and current approaches to the generation of insulin-producing cells from pluripotent sources, such as ESCs and iPSCs, and critically discusses the hurdles to be taken before insulin-secreting surrogate cells derived from these stemcells will be of clinical use in humans.”
Another likely future clinical application of iPSCs is restoration of neural functioning after spinal cord and other diseases which destroy neurons. The October 2011 report Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes relates: “Stem cell-derived motor neurons (MNs) are increasingly utilized for modeling disease in vitro and for developing cellular replacement strategies for spinal cord injury and diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Human embryonic stem cell (hESC) differentiation into MNs, which involves retinoic acid (RA) and activation of the sonic hedgehog (SHH) pathway is inefficient and requires up to 60 days to develop MNs with electrophysiological properties. This prolonged differentiation process has hampered the use of hESCs, in particular for high-throughput screening. We evaluated the MN gene expression profile of RA/SHH-differentiated hESCs to identify rate-limiting factors involved in MN development. Based on this analysis, we developed an adenoviral gene delivery system encoding for MN inducing transcription factors: neurogenin 2 (Ngn2), islet-1 (Isl-1), and LIM/homeobox protein 3 (Lhx3). Strikingly, delivery of these factors induced functional MNs with mature electrophysiological properties, 11-days after gene delivery, with >60-70% efficiency from hESCs and human induced pluripotent stem cells (hiPSCs). This directed programming approach significantly reduces the time required to generate electrophysiologically-active MNs by approximately 30 days in comparison to conventional differentiation techniques. Our results further exemplify the potential to use transcriptional coding for rapid and efficient production of defined cell types from hESCs and hiPSCs.”
Another different potential application of iPSCs is creation of neural stem cells with deadly gene payloads which seek out distributed cancer tumor cells and kill them. The October 2011 publication Glioma gene therapy using inducedpluripotentstem cell derived neural stemcellsreports “Using neural stemcells (NSCs) with tumor tropic migratory capacity to deliver therapeutic genes is an attractive strategy in eliminating metastatic or disseminated tumors. While different methods have been developed to isolate or generate NSCs, it has not been assessed whether inducedpluripotentstem (iPS) cells, a type of pluripotentstemcells that hold great potential for regenerative medicine, can be used as a source for derivation of NSCs with tumor tropism. In this study, we used a conventional lentivirus transduction method to derive iPS cells from primary mouse embryonic fibroblasts and then generated NSCs from the iPS cells. To investigate whether the iPS cell derived NSCs can be used in the treatment of disseminated brain tumors, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected into the cerebral hemisphere contralateral to a tumor inoculation site in a mouse intracranial human glioma xenograft model. We observed that NSCs expressing the suicide gene were, in the presence of ganciclovir, effective in inhibiting the growth of the glioma xenografts and prolonging survival of tumor-bearing mice. Our findings provide evidence for the feasibility of using iPS cell derived NSCs as cellular vehicles for targeted anticancer gene therapy.”
The citations listed here are only a sampler of the many publications relating to iPSCs that appeared in the last few months. Nonetheless, the findings reported in this post and in my June 2011 post are sufficient to allow concluding that significant progress is being made in:
- Increasing efficiency and rapidity of iPS cell reprogramming,
- Achieving freedom from genetic contamination in iPSCs,
- Understanding reprogramming factors and the multiple options that can be used for iPSC reprogramming,
- Finding specific transcription factors that increase reprogramming efficiency, speed and reliability for specific target cell types,
- Achieving consistent pluripotency,
- Controlled targeting of iPSC differentiation,
- Use of iPSCs to create tissues which provide models of specific diseases as well as treatment options,
- Use of iPSCs in drug development,
- iPSC therapies for specific diseases in small-animal models, and
- employing iPSCs for organ regeneration in animal models.
In that June 2011 report on iPSCs, I wrote “My vision of closing the loop in the stem cell supply chain remains as a possibility for the future but many obstacles must be overcome before that possibility matures into reality.” The research reported during the last four months suggests that these obstacles are being vigorously attacked and, in stages, surely overcome. My guess is that clinical use of iPSCs is still 3 or more years away, but that it is assuredly coming and will become very big when it gets here.