The Retrospective Overview on Bioengineering Application of Induced Pluripotent Stem Cell

Min Jae Kim

Keywords: iPS Cell, ES cell, Regenerative Medicine, Bioengineering, Organ Transplant, Stem Cell, Yamanaka Shinya, disease model

Abstract

The creation of the first Induced-Pluripotent Stem cell (iPS cell) from mice fibroblast cells in 2006 was promising. Subsequently in 2007, attempt to induce adult human fibroblasts into IPS cell was successful (Takahashi et al., 2007), thus opening its universal application into regenerative medicine. From this innovation, the continuous studies have allowed the iPS cell technology to develop regenerative medicine, from generating hepatocytes for liver transplant to modeling Alzheimer's Diseases through differentiating specific disease causing genome in the iPS cell (Zhang et al., 2016). However, despite the phenomenal potential, there still remain challenges to generate fully functional organs for universal transplant, such as the spontaneous mutation of the stem cells into teratoma. Nevertheless, the iPS cell is prospective field that became breakthrough in regenerative medicine. This research provides a retrospective view of current IPS cell application in regenerative medicine and its current research obstacles.

Introduction

Originating from the Dr. Yamanaka's successful induction of the pluripotent stem cells from mouse and human fibroblasts in 2006, the iPS cell provided a substantial potential in the regenerative medicine ever since. This specific stem cell was created as an alternative to preexisting ES cells but without severing out the embryo and containing the patient’s genetic information at the same time. As a result, Dr. Yamanaka and his team extracted the common four protein factors embryonic Stem Cell (ES Cell) for somatic cell reprogramming. The outcome was promising and Dr. Yamanaka and Takahashi was able to derived the iPS Cells from the somatic cells (Takahashi et al., 2006). Since derived stem cell is pluripotent, the cell is able to be differentiated into multiple functional cells and tissue structures. Unlike the mesenchymal multipotent-stem cells located in bone marrow stroma, which is only able to produce erythrocytes, leukocytes, thrombocytes, the pluripotent stem cell is to be transformed into various cell types and numbers given specific conditions.

Contrary to the previous Embryonic Stem Cells (ES cells), which are derived from the inner cell mass of fresh or frozen embryos at the blastocyst stage of development (Thomson et al., 1998), iPS cell can be derived from somatic skin cells and still share the similar traits as the ES cells. Due to its accessibility, the iPS cell is hailed as a breakthrough in the regenerative medicine as it is able to proliferate semi-infinitely, providing its eligibility for organ transplant and disease modeling.

Through this discovery, iPS cells have been used in many research and clinical studies including disease modeling, regenerative medicine, and drug discovery/drug cytotoxicity studies. (Singh et al., 2015). This paper provides an overview the bioengineering applications of iPS cells until present from diverse aspects and future challenges ahead of this innovation.

The Three Stem Cells

Embryonic Stem cell Human IPS cell Adult Stem Cells
Stem Cell Type Pluripotent Pluripotent Unipotent/ Multipotent
Proliferation Rate

*The rate is relative to other types of stem cells

Very High Very High Depends on the host tissue
Rejection Response Yes No Rejection No Rejection
Ability to transform to other tissue or cell Very flexible Very flexible Limited
Eligibility for direct transplant Need to modified into specific cells for transplant Need to modified into specific cells for transplant Direct Transplant Possible
Risk of Teratoma Yes Yes No Risk of Teratoma
Life Ethical Problems Yes No Ethical Problems No Ethical Problems

Figure 1. The Comparison between two stem cells.

The Embryonic Stem Cell (ES Cell) is pluripotent cell derived from the blastocyst of an embryo, which the first human ES Cell was manufactured by Dr. Thomson in 1998. The blastocysts are obtained from in vitro fertilization and that later is enlarged through cell culturing (Thomson et al., 1998). The ES Cell has a potential to be induced into other cell forms, but must be differentiated equally to the host tissue for compatibility. The ES cells can also form teratoma to the host body, a tumor with organ components. Furthermore, the ES cell contains high risk of transplant rejection since the genetic information of the ES Cell is not compatible with the adjacent host cells, thus provoking immune response.

The Induced- Pluripotent Cell (iPS Cell) shares the similar qualities as those of ES Cell: the pluripotent nature with fast proliferation rate. One study suggests the proliferation rate correlates with the stem cell’s reprogramming efficiency, so that pluripotent stem cells (ES cell and iPS cell) are highly proliferative compared to other multipotent or unipotent stem cells (Ruiz et al., 2011). The major differences between the two stem cells are the type of cells used for derivation and the compatibility. Contrary to the Embryonic Blastocysts, the iPS cells are feasible to be derived from normal somatic cells. Another difference is that iPS cells are very compatible to the host body. Since iPS cells is derived from a host’s own somatic cells, the genetic information the stem cell possesses is equal to the hosts’, thus making the transplant compatible. Yet the iPS cell is also likely to stimulate teratoma as the ES cell does.  

On the other hand, the adult stem cell is not pluripotent, but rather is able to be differentiated into predetermined somatic cells. These stem cell types include hematopoietic stem cells, bone marrow stromal stem cells, mesenchymal stem cells, which all are induced to specific cells for metabolism facilitation (National Institute of Health, 2015). Thus, adult stem cell has limited range for cell transformation. However, different from the ES cells and iPS cells, the adult stem cell has a relatively low rejection possibility and can directly be transplanted. One instance of its application is the hematopoietic stem cell transplant for thalassemia patients (Lucarelli et al., 2012). The stem cells are transplanted directly and require no secondary procedure for differentiations, with no risk for ethical problems nor teratoma formation, since there is no genetic alteration process involved.  

[Supplementary information on teratoma is on Future Challenges for iPS Cell Bioengineering Application]

Formation of the iPS cell

The iPS Cell is created by inserting several protein factors such as Oct3/4, Sox2, Klf4, and c-Myc into the somatic cell to reprogram it as a stem cell (Takahashi et al., 2007). These four factors (Yamanaka Factors) are based on the 24 essential factors common in the ES Cell. Additional factor configuration, Oct 4, Sox 2, NANOG, Lin 28, was introduced as feasible alternative for deriving the cell (Yu et al., 2007).

The reprogramming into iPS cell include inserting the four factors into the somatic cells. The factors are first must be inserted through the retroviral vectors in RNA form. Then, the retrovirus inserts the factor into the nucleus of the somatic cell (Figure 2) Then the nucleus reprograms itself and the somatic cell transforms into an iPS cell. After the completion of iPS cell derivation, the cells are then cultured in the similar condition as ES cell culturing, by providing feeder cells to enhance cell proliferation and colony formation (Takahashi et al., 2007). The cell bodies then can be used for clinical purposes in forms of transplant and disease modeling.

 

Figure 2: The Derivation Process of iPS Cell

The Application of iPS Cell on Regenerative Medicine

The regenerative medicine has expanded greatly toward the application of iPS cells. One of the major applications of the iPS cell is transplantation in regenerative medicine. Due to its potential for differentiation into variety of cells and its semi-infinite proliferation in colonies, the iPS cell is very suitable for transplantation. The major cell types for transplants are hepatocyte, cardiomyocyte, neuron that are possible to from livers, heart grafts, and nerve fibers.

  • Hepatocytes

The hepatocyte is one of the most researched cell for regeneration due to its rapid growth process after the resection of more than 50% of its mass (Fausto and Campbell, 2003). Also, due to liver’s crucial effect of detoxification and nutrition storage in human metabolism, the hepatocyte transplant is more accessible and pivotal in regenerative medicine.

There are multiple trials that succeeded in forming hepatocytes or tissues exhibiting similar functions as those of a liver. Espejel studies proved that the iPS cells were intrinsically able to differentiate into mature hepatocytes. The resulting iPS cell-derived hepatocytes had similar proliferative capabilities of normal hepatocytes and were able to regenerate the liver after transplantation and two-thirds partial hepatectomy (Espejel et al., 2010).

The first functional organ (liver) differentiation from the human pluripotent cell was in 2013. The implanted liver bud onto the iPS Cell derived from human somatic cells (iPSC-LB), the cell transplants became functional by connecting to the host vessels. In addition, this vascularized tissue performed liver-specific functions such as protein production and human-specific drug metabolism (Takebe et al., 2013). Another attempt to differentiate the hepatocyte, from the human hair follicles (HF- MSC)- derived iPS cell, were unable perform full generation. However, the cell lines were able to induce a generation of hepatocyte-like cells (HLC) that expressed hepatocyte markers and drug metabolism-related gene (Shi et al., 2016).

  • Cardiomyocytes

The development of iPS cell into cadiomyocytes is also promising. Contrary to hepatocytes, the stem cell itself must have capacity to differentiate into various phenotypes (nodal, atrial, and ventricular) and responsive to electric stimulation. But in 2009, a research from Zhang and his colleagues successfully generated functional cardiomyocytes from a human iPS. They concluded that the iPS cell are a viable option in cardiac repair, as the differentiated cardiomyocytes exhibited responsiveness to ?-adrenergic stimulation manifest by an increase in spontaneous rate and a decrease in action potential duration (Zhang et al., 2009).

Progressing from the previous research, the iPS cell- differentiated cardiac progenitor cell has been developed. The initial study of Zakharova have shown that the transplantation in the infarcted heart allowed the rat and human progenitor cell sheet allowed to differentiate into cardiomyocytes and improve cardiac contractile function (Zakharova et al., 2010). The additional studies by Carpenter and his colleagues suggests that implanted iPS cell-derived cardiac progenitor cells were successful in differentiating into optimal cardiomyocyte, thus showing improvement in infarcted heart (Carpenter, 2012). Another research confirmed a significant condition for a successful cardiomyocyte transplant. The result suggests that the difference of days (4, 8, 20, 30 days) before iPS cell differentiation into cardiomyocyte determines the engraftment ratio. The result yielded the 20 day cardiomyocytes had the highest engraftment efficiency with improvement by cell therapy (Funakoshi et al., 2016).

Due to previous studies on potential cell therapy with differentiated cardiomyocytes, one applicatory field of the iPS cell is the treatment of the cardiovascular rupture through heart graft bypass and cardiomyocyte transplant. One approach is for the stem cell to differentiate into cardiomyocyte cells in repopulate the decellularized human heart ventricles. A trial to create a cardiomyocyte-like cells (CLCs) for heart graft was successful through rapid production of human induced pluripotent stem cells in total of 24 days with the use of Transcription activator-like effector nucleases (TALENS) (Garreta et al., 2016). The cultured CLC were then used to decellularized acellular myocardial scaffold and displayed significant increases in the levels different ion channels determinant for calcium homeostasis and heart contractile function.

  • Neuron and Disease Modeling

One of the most significant areas for iPS cell transplant is in neural regeneration. For both neurodegenerative diseases and trauma, the pluripotent cell is optimal for transplant in the lesion and regenerate the neuron stem. Several approaches for neural regeneration are held, such as Induction of the neural progenitor cell for transplant. A research held by Fukusumi and her colleagues concluded that neural lineage cells can be derived from most human iPS cell clones and induced- neural progenitor cells exhibiting mid/hindbrain-type property (Fukusumi et al., 2016), thus suggesting the prospective application of iPS cell in neural cell therapy.

Within the diversified field of neural regeneration, the treatment of the neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Huntington’s disease (HD) is closely related to the iPS cell therapy. One study from Wernig and colleagues show that mouse fibroblast derived iPS cells formed clusters of neuroepithelial-like cells in media (Wernig et al., 2008). The differentiated cells expressed the neural stem cell markers nestin, Sox2 and Brn2 can be efficiently differentiated into neural precursor cells, which they were transplanted in fetal mouse brain and migrate into various brain regions where they differentiate into glia and neurons (Wijeyekoon and Barker, 2008). Furthermore, the transplanted iPS Cells were induced to differentiate into dopamine neurons and improve behavior in a rat model of Parkinson's disease (Wernig et al., 2008). Thus, as cell therapy on neurodegenerative diseases were proven effective, there has been an increasing interest for the regenerative medicine without using the patient’s own cells, but rather using the iPS cell stocks established from the donor somatic cells. The cells must be homozygous at human leukocyte antigen (HLA) with patient's HLA type for compatibility (Okano and Yamanaka, 2014). Hence, these “cell banks” are pragmatic for rapid transplantation and significantly reduce graft rejection reactions through HLA matching. (Supplementary information in Current Challenges for iPS Cell Bioengineering Application).

Furthermore, iPS cell not only favorable in regenerative transplant, but also effective in disease modeling and possibly the cure for neurological disorders. The patient-specific iPSC-derived neural cells can recapitulate the phenotypes and genes of the specific neurological disorders, which when studied, would expand our knowledge in the disorder pathogenesis, mechanisms, and develop screening platforms (Russo et al., 2015). The research held by Nekrasov and colleagues was to explore the HD pathology of the derived neurons. Since HD is a neurological disorder that prompts loss of GABAergic medium spiny (GABA MS) neurons, the researcher's intention was study the pathology from the iPS - derived GABA MS-like neurons (GMFLNSs). The induced neurons recapitulated disease pathology as mutant protein aggregation, increased number of lysosomes, and enhanced neuronal death during cell aging (Nekrasov et al., 2016).  For instance, Alzheimer's disease (AD) is the dementia characterized by deposition of extracellular ?-amyloid plaques, intracellular neurofibrillary tangles, and extensive neuron loss. There are currently three causative genes (APP, PSEN1, and PSEN2) that cause mutations (Zhang et al., 2016) and applicable as signal markers. A study from Takuya and colleagues suggests that early-onset familial AD (FAD) patient cell- derived iPS cell differentiated into neurons that have increased amyloid ?42 (A?42) secretion that recapitulated the molecular pathogenesis of mutant presenilins of the patients. The research has also proven that the ?-secretase inhibitors are effective in suppressing of A?42 and A?40 production in the iPS-derived neurons, thus providing the prospect for drug candidates (Yagi et al., 2011).

Future regenerative medicine of iPS cell Applications (Organ Formation)

Despite the numerous cases of successful transplant of functional cells and disease modeling, there are various fields in which the iPS cell differentiation is still studied. In addition to previous breakthrough, renal, corneal, muscular, cartilage, and lung regeneration are currently studied. Similar to neurological disease modeling, the chronic diseases on each organ are feasible for modeling. The iPS cells from patients can be used as a disease model for enhanced comprehension of pathophysiology and develop treatment (Freedman and Steinman, 2016).

The induction of the respiratory epithelial cells from the patient-derived iPS cell has been successful. Ghaedi and colleagues generated an alveolar epithelial type 1 (AETI) and type 2 (AETII). The two types of epithelial cells contained sufficient amount of chemical markers and similar phenotypes with the mature AETII and AETI, such as surfactant protein C, mucin-1, surfactant protein B, and epithelial marker CD54 (Ghaedi et al., 2013).  Furthermore, the cultured cells were able to proliferated in lung tissue scaffold, thus providing the potential application of iPS-derived lung tissues for patients with diverse respiratory inflammatory diseases and pulmonary infarction.

Another exceptional function of iPS Cell regenerative medicine is in the orthopedic transplant. Especially cartilage and chondrocytes regeneration are extremely beneficial for patients suffering from degenerative joint diseases, such as osteoarthritis. Similar to generating neurons through inducing neural progenitor cells, the chondrocytes are formed by mesenchymal stem cells (MSCs), which are capable in generating diverse cell types such as endothelial cells, smooth muscle cells, bone, cartilage, and lipid cells. Thus, the method for cartilage regeneration is by iPS cell differentiating itself to mesenchymal stem cell. Lian and colleagues suggest that this approach was highly satisfactory, as the human iPS cell derived MSC (IPSC-MSC) displayed a higher proliferative capacity than bone marrow-derived MSCs. With approximately 20 days of iPS differentiation, the generated IPSC-MSC expressed MSCs surface markers and undergoes differentiation process, such as adipogenesis, osteogenesis, and chondrogenesis (Lian et al., 2016). However, there remain several challenges to perfect the regenerated cartilage transplant. The chondrocytes cell culture includes additional input such as condense cell bodies and pellet cultures. This co-culture conditions between feeder cells and external bodies provide high risk for contamination of differentiated cells (Lietman, 2016).

Future Challenges for iPS Cell Bioengineering Application

Despite the tremendous potential the iPS cell technology possess, there are still overall challenges to resolve such as reducing chances of erratic transformation of iPS cell colony into teratoma and its pharmatheutical efficiency.

The tumorigenesis of the iPS cell is primarily known as the two factors that are imputed for stem cell derivation process, c-Myc and Klf4. As the factors are enveloped within the retrovirus when they are inserted into the cell nucleus, the reprogramming process through the retroviral input increases the risk of tumorigenesis and alters the gene expression into becoming tumor cells (Lanctot, 2011). Similar to stem cells, tumor cells perform rapid proliferation with semi-infinite cell divisions. One study by Gutierrez-Aranda and colleagues suggests that there were no site-specific differences in teratoma composition at histological level, and the rate of teratoma formation on mice model was 100%, regardless the type of injection (Gutierrez-Aranda, 2010). Thus, the high risk of tumorigenesis is still an obstacle for iPS cell- derived organs for human transplantation.

Considering that the compatibility and tumorigenesis are resolved, the efficiency of iPS cell for pharmaceutical use and cell therapy is yet still low. The four factors inserted for programming of the cells are known as minimum necessary gene input that sustain cell structure and successful differentiation. However, the process takes a long time (3~4 weeks for human iPS Cell derivation) and has one in hundred chance. There have been several attempts to facilitate the derivation process with faster pace. A study of Hochedlinger and his research team at Harvard Stem Cell Institute sought the best combinations of factors for most successful iPS cell yield. The addition of ascorbic acid (Vitamin C) and CHIR-9021 into original four factors provided with increased effectiveness of iPS cell formation. Also, the yield time was only 48 hours to form a colony, which issues a prospective step toward the stem cells’ practical application (Harvard Stem Cell Institute, 2014). Another alternative is the “cell banks” that store iPS cells with various HLA types for transplant compatibility. The cell banks are now researched such as New York Stem Cell Foundation (NYSCF) and Coriell Institute in Camden, New Jersey (Grens, 2014). Furthermore, The European Bank for induced pluripotent cells (EBiSC) was already established in February 2, 014 and currently in operation. The EBiSC expects a capacity of 10,000 cells and processes about 1,000 lines per year. However, there still remain several questions on the manufacturing and the management of the stem cell market in the future.

The iPS cell technology has been expanding ever since from its discovery in 2006. The extensive researches on differentiating iPS cells into diverse categories of human cells have been successful. Our next challenge is to utilize these studies to generate full complex organs as complete cell therapy and bioengineering. Substantially, the iPS cell technology is hallmark in the regenerative medicine and provides near infinite application not only as transplantation, but also in disease model and drug discovery. Thus, this stem cell has a massive potential in revolutionizing the medical field, as the current challenges are resolved.

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