Induced pluripotent stem cells (iPSCs) can be generated by reprogramming of adult/somatic cells. The somatic cell reprogramming technology offers a promising strategy for patient-specific cardiac regenerative medicine, disease modeling, and drug discovery. iPSCs are an ideal potential option for an autologous cell source, as compared to other stem/progenitor cells, because they can be propagated indefinitely and are able to generate a large number of functional cardiovascular cells. However, there are concerns about the specificity, efficiency, immunogenicity, and safety of iPSCs which are major challenges in current translational studies. In order to bring iPSC technology closer to clinical use, fundamental changes in this technique are required to ensure that therapeutic progenies are functional and nontumorigenic. It is therefore critical to understand and investigate the biology, genetic, and epigenetic mechanisms of iPSCs generation and differentiation. In this spotlight paper the discovery, history, and relative mechanisms of iPSC generation are summarized. The current technological improvements and potential applications are highlighted along with the important challenges and perspectives. Finally, emerging technologies are presented in which improvements to iPSC generation and differentiation approaches might warrant further investigation, such as integration-free approaches, direct reprogramming, and the development of iPSC banking.
Myocardial infarction (MI) is an important manifestation of coronary artery disease (CAD) and major cause of death and disability worldwide. MI occurs when prolonged ischemia irreversibly destroys distal blood vessels and myocardium, causing apoptosis or cell death, eventually triggering cardiac remodeling or sudden death [
Regenerative therapies offer great promise for patients with heart disease by using angiomyogenesis to create a source of replacement for lost or damaged cardiac tissues associated with MI [
First seen in studies of experimental embryology, mouse or human embryonic stem cells (ESCs) were discovered as a kind of pluripotent stem cells derived from the inner cell mass of a developing blastocyst. A great milestone in stem cell biology occurred when it was found that these cells could be propagated in culture in an undifferentiated state [
The technique of somatic cell nuclear transfer (SCNT) was established during the 1950s to probe the developmental potential of nuclei by transplanting them into enucleated oocytes [
Initially, Yamanaka showed that stem cells with properties similar to ESCs could be generated from mouse or human fibroblasts by simultaneously introducing four TFs [
In order to efficiently produce iPSCs as research tools, and ultimately translate laboratory results into clinical applications, a number of different somatic cell reprogramming approaches have been developed, including gene transduction and integration-free methods (Figure
The current approaches of iPSC generation. There are a number of different approaches for iPSC generation from somatic cell reprogramming, including gene transduction and integration-free methods. Retroviral or lentiviral gene transduction is the most efficient and widely used method, but the integration of TFs into a host genome increases the concerns of oncogenicity and mutagenesis. The integration-free methods include using nonintegrating virus, plasmids, recombinant proteins, synthesized mRNA, microRNAs, and small molecules. Most of these approaches are safe, but reprogramming efficiency is lower than that of gene integration. The activation of endogenous pluripotency is considered an important criterion of high-quality fully reprogrammed iPSCs.
Gene transduction can be used to generate iPSCs using retroviral or lentiviral approaches, which are currently the most efficient and widely used methods, although progress is rapidly growing in the use of other gene delivery methods. Twenty four pluripotency-associated candidate genes were initially evaluated in an assay system by retroviral transduction in order to identify the effective reprogramming factors. After successive elimination of individual factors, the minimal four genes comprising Oct4, Klf4, Sox2, and c-Myc (OSKM) were determined to induce reprogramming of mouse embryonic fibroblasts (MEFs) [
As the reprogramming approaches rise in number, it is important to generate optimal reprogramming factor cocktails for various adult cells. Each protocol must take into consideration the reprogramming efficiency because currently it is at a very low level in the majority of current studies (typically much less than 1% [
Of great concern in these protocols is safety, as many reprogramming factors can be linked with the pathophysiology of cancer, such as c-Myc which is a very strong proto-oncogene [
In a different example, a detailed protocol was developed to derivate iPSCs from cord blood stem cells using retroviral transduction with only two factors (Oct4 and Sox2) that are a prerequisite in the majority of present studies [
As discussed above, the low efficiency of iPSC generation is a great challenge for the study of the molecular processes or mechanisms during the early phase of reprogramming. The use of an inducible transduction system, which can be flexibly controlled by the inert drug tetracycline (Tet) or doxycycline (Dox), allows for increasing efficiency and the selection of fully reprogrammed iPSCs [
The inducible transgene and excisable system for iPSC generation. (a) The inducible transgene system conta ins two main components, reverse-tetracycline-dependent transactivator (rtTA) and the inducible promoter controlling the expression of reprogramming genes (OSKM). In the absence of doxycycline (Dox) induction, the specific promoter lacks binding sites for endogenous TFs, so it is virtually silent. In the presence Dox, the transactivator binds tightly and specifically to the promoter and activates transcription of OSKM genes. (b) The primary somatic cells (such as fibroblasts) were infected with inducible lentivirus encoding OSKM to generate “primary” iPSCs (1-iPSCs) with Dox. These were then injected into blastocysts to create chimeras or formed into embryoid bodies for the generation of “secondary” somatic cells. These then carry the inducible reprogramming factors, which were isolated and used to generate secondary iPSCs (2-iPSCs) using Dox induction. (c) The schematic of integration-free iPSCs generated by the transposon and
To generate genetically homogeneous cell populations, primary somatic cells (such as hepatic cells, hematopoietic cells, or fibroblasts) can be infected with Dox-inducible lentivirus encoding OSKM to generate the “primary” iPS cell lines with Dox in culture medium [
Current studies are seeking an even more ideal resource of “secondary” somatic cells. Using “primary” iPSCs generated from primary human fibroblasts and keratinocytes with the Dox-inducible system, cells can be differentiated into fibroblast-like cells as the “secondary” somatic cells (Figure
Unfortunately retroviral transgenes are epigenetically silenced towards the end of reprogramming [
Recently, double transgenic reprogrammable mouse systems have been developed as the most advanced techniques in mouse inducible iPSC generation. After a generation of “primary” iPS cells are generated using the inducible system, cells were used to breed chimeric mice whose somatic cells could be isolated and reprogrammed
Current retroviral or lentiviral reprogramming strategies are utilizing the integration of TFs into host genome, increasing concerns of oncogenicity and mutagenesis [
Using replication-defective adenoviral vectors, the first integration-free iPSCs were generated from fibroblasts and liver cells using adenoviruses transiently expressing OSKM [
In order to eliminate the risk of oncogenicity in the integration protocols, several laboratories have refined the process by using “excisable” methods, such as transposon systems and
A more attractive approach allowed for iPSC generation using transfection of mRNAs coding for the reprogramming factors into human primary somatic cells [
The above studies support the theory that transient expression of the exogenous reprogramming factors is sufficient to induce pluripotency in somatic cells. They also do not require genomic integration and can be eliminated after transfection, giving them an independence that is considered an important criterion of high-quality fully reprogrammed iPSCs. Recently, other DNA-free methods with more convenient manipulations are in development to activate the endogenous pluripotent genes for reprogramming such as microRNAs, recombinant protein, and small molecules.
It is first required to understand the molecular changes underlying iPSC derivation for the development of alternative and safer strategies for reprogramming. Gene expression profiling studies in fibroblasts have revealed three phases of reprogramming termed initiation, maturation, and stabilization [
Recombinant protein delivery represents a straightforward reprogramming strategy. The first protein-induced pluripotent stem cells (piPSCs) generated using these methods were reported by two independent research groups [
In order to improve the efficiency of nonintegrating reprogramming approaches, several screens for chemical compounds have been performed. A number of small molecules have been identified since, that modulate the induction of reprogramming while significantly improving the efficiency and quality of iPSC generation and functionally replacing exogenous OSKM. A single chemical compound is not able to entirely replace the function of a reprogramming factor in current studies but it can work with other modulators. Named on the basis of their reprogramming mechanisms, they can be classified into epigenetic modifiers, signaling pathway modulators, cell senescence alleviators, and metabolism regulators [
Epigenetic modifiers are used most commonly in various iPSC generation protocols because the epigenetic changes (e.g., DNA demethylation and histone acetylation) of pluripotency genes are the key feature of reprogramming. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was initially reported to effectively reprogram primary human fibroblasts without introduction of the oncogene c-Myc [
In addition to epigenome remodeling, signal transduction pathways mediated by extrinsic factors and intrinsic transcriptional networks cooperate to reprogram somatic cells into iPSCs [
Organismal aging or senescence was identified as another one of the barriers for iPSC reprogramming, resulting in slow kinetics and low efficiency of processes [
iPSC generation requires energy transition from mitochondrial oxidative phosphorylation to perform glycolytic metabolism [
Remarkably, after the screening of nearly 10,000 small molecules, seven molecule compounds (VPA, CHIR99021, Tranylcypromine, 616452, FSK, and DZNep) were found, reprogramming mouse fibroblasts into iPSCs solely, obtaining dozens of iPSC colonies per 50,000 cells [
In addition to pluripotent TFs, endogenous specific microRNAs (miRs) have been reported to be highly expressed in ESCs, termed ESC-specific miRNAs [
The transfection of miR-302-family, for instance, was reported to reprogram several human cancer cell lines into an ESC-like pluripotent state, with similar gene expression patterns and differentiation functions [
The inhibition of microRNAs enriched in somatic cells may also facilitate the reprogramming procedure. For example, loss of miR-145 impaired differentiation and elevated its direct target genes, including Oct4, Sox2, and Klf4, but its increasing expression inhibited ESC self-renewal, repressed pluripotent genes, and induced lineage-restricted differentiation [
The somatic cell reprogramming and iPSC generation approach have only recently become a popular target for regenerative medicine research, but there has been tremendous enthusiasm in translational studies of iPSCs within every field of medicine. Specific differentiation into the 3 primary germ layers and all the cell types potentially provides many benefits for cell-based therapies, disease modeling, and drug development [
Various cell delivery approaches have been developed to enhance stem/progenitor cell engraftment, survival, and integration to host tissues after MI and have been performed in multiple clinical trials of cardiac regenerative therapy, as summarized in our review [
Patient specific or autologous iPSC-derived cells will play an increasingly important role for replacing lost or damaged tissues [
There are 3 major approaches for differentiation of human iPSCs to cardiomyocytes including embryoid body (EB) [
The aptitude of iPSCs for
The cardiomyocytes derived from iPSC or ESCs have been shown to improve cardiac functions after transplantation, but the functional integration of grafts into injured heart tissue has not been demonstrated. The transplantation of ESC-derived cardiomyocytes in uninjured heart had consistent 1 : 1 host-graft coupling, while the grafts in injured hearts were more heterogeneous and typically included both coupled and uncoupled regions [
In addition to cardiomyocytes, other cardiovascular cell lineages, such as endothelial cells (ECs) and smooth muscle cells, play an important role in the functional restoration of MI [
It has become clear that cell therapies combined with tissue engineering techniques (such as myocardial cell sheets) can increase stem cell survival and retention, thereby enhancing therapeutic effects [
Transdifferentiation is a new paradigm that has been devised to generate cardiovascular lineage-specific precursor cells directly from somatic cells (Figure
The model of direct reprogramming of fibroblasts into cardiomyocytes in infarct heart. The transient overexpression of cardiac specific TFs, GMT, is able to generate cardiac lineage-specific cells directly from somatic cells. This process “skipping pluripotency” does not require transition through a pluripotent intermediate, immensely eliminating the risk of tumorigenicity.
Direct myocardial reprogramming continues to be explored under
However, the GMT overexpression in murine tail-tip fibroblasts and cardiac fibroblasts is an inefficient method to induce direct myocardial reprogramming, and lacks the molecular and electrophysiological phenotypes of mature cardiomyocytes [
Interestingly, pretreatment of VEGF to infarcted myocardium was reported to enhance the efficacy of the GMT-mediated reprogramming strategy, improving myocardial function and reducing the extent of myocardial fibrosis [
“Skipping pluripotency” is an alternative strategy for the production of functional endothelial cells directly induced from somatic cells. This reprogramming approach mediated by epigenetic mechanisms does not require transition through a pluripotent intermediate during the process of transdifferentiation, immensely eliminating the risk of tumorigenicity. This provides a new line of thought for the generation of lineage-specific cell types derived from somatic cells and may facilitate the potential applications of reprogramming in cardiac regenerative therapy. In one study, human fibroblasts were induced to generate partial-iPSC for 4 days by transfecting OSKM into the nuclei and clearly displayed the potential to differentiate into endothelial cells in response to defined media and culture conditions [
Alternatively, based on the potential of miRNAs regulating developmental and reprogramming processes, a combination of miRNAs was identified to induce direct reprogramming of fibroblasts to cardiomyocyte-like cells
In addition to their regenerative capacity in cell therapy, iPSC-derived myocardium provides an important
The potential applications and challenges of iPSCs in cardiac regenerative medicine. iPSCs are a cell therapy strategy for replacing lost or damaged cardiac tissues. They can be generated from patient specific somatic cells and are capable of generating large numbers of unambiguous cardiomyocytes using defined protocols. The major challenges in the current translational studies are concerns about the specificity of function maturation, low efficiency, immunogenicity, and safety of iPSCs. In addition to their applications in cell therapy, iPSC-derived cardiovascular cells provide an important
The application of human iPSC-derived cell types can accurately recapitulate relevant human diseases from animal models. Stem cells are available in essentially unlimited quantities and exhibit a more stable phenotype in long-term culture, which is superior to primary cells. The human specific iPSCs also can also be differentiated into any human cell type and early efforts have focused on four main areas: neurons, hepatocytes, cardiomyocytes, and pancreatic beta islet cells, with widely known molecular and biochemical signals driving
The first disease-specific iPSC line was generated from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS) and it was successfully differentiated into motor neurons which were destroyed in ALS [
Applications of iPSCs for modeling diseases
In the study of cardiovascular diseases, human iPSC-derived cardiomyocytes can serve as a cardiac model to be used for diverse basic studies ranging from cellular electrophysiology to biochemistry [
In addition to the investigation of the mechanisms of human diseases, iPSC-derived cell models also play an important role in the evaluation of the potential therapeutic efficacy of drugs or small molecules. In this regard, access to abundant populations of human cardiomyocytes is of particular interest to the pharmaceutical industry as a tool to develop new cardioactive compounds for correcting cardiac disease phenotypes and for screening the potential cardiotoxicity of new compounds.
One such study employed the use of cardiomyocytes differentiated from patients with arrhythmogenic disorders. Patient-specific iPSCs were used as a guide for assessing drug therapeutic effects and tailoring medical treatment for patients [
Finally, iPSC can be used for the identification of novel target genes that play a potential role in the development of therapies for myocardial diseases. When molecular mechanisms of relevant diseases are recapitulated, the identification of gene candidates could be used for target-agnostic drug screening. RNA interference (RNAi) technology has been applied on a genome-wide scale for several biological processes to identify the potential new targets effectively [
Adverse reactions and drug toxicity represent major challenges for pharmaceutical industries, contributing to the high cost of drug development. Thus, it is important to develop the predictive human cellular systems for the complementation of current toxicity tests to establish routine screening for toxicity pathways [
Although the discovery of iPSCs is encouraging and has brought forward numerous successful studies, it is just beginning to be used in the preclinical phase of human trials and the potential is still emerging [
Given the successes of iPSC transplantation experiments in animal models, clinical trials will be taking place in the near future [
Current progress has contributed to an in-depth investigation of pluripotency mechanisms that are part of the processes of ESC or iPSC generation. Pluripotency reporter models such as Oct4 promoter and GFP transgene mouse [
One major pitfall is the tendency for iPSCs to form teratomas and current differentiation protocols cannot completely eliminate residual undifferentiated cells. This problem can be improved by morphological selection, label or staining strategies using fluorescence activated cell sorters (FACS), and drug selection approaches [
Although the purity of these isolation protocols was reported up to ~99%, it is still difficult to remove the undifferentiated cells and the differentiated noncardiac cells completely to obtain entirely purified derived cardiovascular cells. Even if very few native iPSCs (included differentiated) are left over, the pluripotent residual and unwanted populations can proliferate after implantation
It is advantageous for iPSCs or ESCs to be propagated indefinitely
Although some laboratories describe successful variable cell differentiation approaches, the conversion of pluripotent cells to terminally differentiated cells (such as cardiomyocytes) is initially inefficient and not readily transferable across various cell lines. It is also very difficult to find the derivatives of many mature cell types. These limitations must be overcome in order to obtain high quality generation of
Scientists continue to refine current protocols and improve cardiac differentiation and maturation of iPSCs, combining optimal devices, culture conditions, materials, and timing [
Before any clinical applications are feasible, the translational studies of stem/progenitor cell based therapy must be able to ensure that the progenies are stable (no differentiation), functional (therapeutic recovery), and safe (no tumorigenicity). Grafts should not elicit severe immune responses of any sort that could threaten the survival of donor cells after transplantation. The probability of immunogenicity associated with the transplantation of stem/progenitor cells and their derivatives has not been clearly addressed and remains one of the greatest obstacles to clinical applications [
The iPSC-based autologous methods can avoid the issues of immunogenicity or immunological rejection, while the allogeneic iPSCs can induce severe immune reactions. Autologous iPSCs generation is often associated with high medical costs and longer hospital duration using the current methods as discussed above. The rapidly effective treatment of some disorders, such as acute MI and spinal cord injury, cannot be achieved within the necessary time frame and it is unrealistic to generate autologous iPSCs from the patient’s biopsy during the ongoing surgery. Although the healthy autologous iPSCs can be collected and stored in “private cell banking” and serve as primary sources for future use, it will be problematic to achieve this on a large scale due to relatively inefficient reprogramming techniques and high costs [
Contributions from multiple studies have guided iPSC research from their discovery to theory and methods of generation, as well as future directions in clinical use. These studies indicate that a network of TFs plays a central role in the maintenance of pluripotency and self-renewal of pluripotent stem cells. The reprogramming of somatic cells back into the embryonic state with pluripotency was realized by overexpressing four pluripotent factors. Most laboratories can now expand various iPSCs robustly using commercially available products according to established protocols. There has since been tremendous enthusiasm in studies of iPSCs within almost every field of medicine. However, there are many limitations for retroviral reprogramming approaches. Many nonintegrating strategies are being developed for the reprogramming process, and the chemical reprogramming protocols can be successful with a combination of different compounds, providing new insight into the minimal or alternate requirements for pluripotent TFs. Studies on the mechanisms of pluripotency induction indicate that all the reprogramming factors are interchangeable. This is a very important consideration in that the manipulation and improvement of iPSC generation is feasible and flexible with an ultimate goal of activation of endogenous pluripotent networks. All of these approaches need to be optimized including gene delivery methods, growth conditions, culture timing, and target cell selection in order to increasing the reprogramming efficiency.
Compared to other stem/progenitor cells, iPSCs can be propagated indefinitely and are able to generate large numbers of functional cardiomyocytes with defined induction protocols. However, prolonged expansion and culture should be avoided due to the increasing risk of chromosomal abnormalities. Derived cardiovascular cells are emerging as an ideal potential option for an autologous cell source for cardiac regenerative therapy. Importantly, a reprogramming strategy that “skips pluripotency” has been developed for the production of functional cell types directly induced from somatic cells under defined conditions. It does not require transition through a pluripotent intermediate and virtually eliminates the risk of tumorigenicity by mediating epigenetic mechanisms. This approach of
In addition to their applications in cell therapy, iPSC-derived cardiovascular cells provide an important
The author declares that there is no conflict of interests regarding the publication of this paper.
This work was supported by NIH Grants, HL089824, HL107957, and HL110740 (Y. Wang).