The principles of this method involve in vitro removal of oocyte nucleus followed by its replacement with donor somatic nucleus. Then, cell division is stimulated by chemicals or electricity up to blastocyst stage. At this stage, cellular mass is isolated and cultured. The resulting embryonic stem (ES) cells are immunologically very identical to donor cells, and no immunosuppressant is required after transplantation to prevent their rejection. However, mitochondrial DNA from maternal oocytes could be potentially immunogenic (9). Major limitations of this method for clinical application are ethical concerns related to germ cell manipulation, chromosomal disorders in derived stem cells, low efficiency of transfer technique, and insufficient supply of human oocytes (8).

An advanced method of cell fusion was developed by Cowan et al. (10) which reprogrammed human normal diploid fibroblasts into ES cells. In this method, human embryonic cells were fused with human fibroblasts, resulting in hybrid cells with stable tetraploid DNA. Characteristics of these cells were similar to human ES cells. However, before clinical application, a set of technical limitations should be resolved. The most important challenge is to abolish ES-like cells after cell fusion.

Different cell extracts can alter gene expression profile in somatic cells (11). Data obtained from experiments on 293T cells, an embryonic kidney cell line, have revealed that extracts of ES cells or embryonic carcinoma cells can induce ES cell phenotype and expression of pluripotency genes. Expression of somatic gene markers such as lamin A was reduced after this manipulation. Besides, these cells gained the ability to differentiate into mesoderm and ectoderm lineages (12). Bru et al. (13) also reported the elevation in expression of pluripotency genes including Oct3/4, Sox2, Klf-4, and c-Myc after exposure of mouse ES cell extracts to 293T cells for 48 h. However, these cells are generally limited in pluripotency potential.

In 2006, the discovery of somatic cell reprogramming to induced pluripotent stem cells (iPSCs) led to a revolution in regenerative medicine (14). iPSCs are basically patient-specific pluripotent cells that are produced by inserting four genes, including Oct4, Sox2, Kfl4, and c-Myc, necessary for fibroblasts to evolve ES-like properties. Recent studies have indicated that only the presence of Oct4 gene may be sufficient to induce pluripotency in adult cells (15). In vitro, iPSCs have efficiently been used for liver tissue construction (15). Takebe et al. (16) showed that co-culture of human iPSCs-derived hepatic endoderm cells with human umbilical vein endothelial cells and human mesenchymal stem cells leads to the formation of liver buds (LBs) in 3D culture condition. Furthermore, iPSCs–LBs injection to mouse resulted in dynamic vascularization. Therefore, these cells can potentially be applied in vast areas including disease modeling, tissue engineering, and drug discovery (17, 18).

To date, therapies based on human ES cells are associated with controversial issues related to ethical concerns in using human embryos and potential risk of immune-mediated tissue rejection. Utilization of patient’s cells in order to avoid ethical concerns and rejection complications is possible by cellular reprogramming, particularly iPSCs technology (19). Based on the present protocols, fibroblasts with skin biopsy origin can be returned to pluripotent stage and serve as a renewable and autologous cellular source (20). However, the original mutation that causes disease will be present in patient-derived pluripotent stem cells. Precise correction of mutation is possible by gene-editing technique, “clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system,” which evolutionarily serves as an immune system in bacteria and archaea against virus and plasmid invasion (Figure 2). The specificity of this technique mainly depends on a guide RNA (gRNA) that can be readily reprogrammed to loci of target gene (21). Editing mutations in iPSCs derived from patients with retinitis pigmentosa was recently used through CRISPR/Cas9 approach (19), which opens a promising era in regenerative medicine and genome engineering.

Hepatic regeneration is a complicated process regulated by growth factors, cytokines, transcription factors, hormones, microRNAs, metabolic pathways, and products of oxidative stress (22). The use of a specific pool of cytokines in a serum-free medium is a prerequisite for liver organogenesis step in differentiation process (23). For example, high doses of activin A are widely used for endodermal induction in human pluripotent stem cells (24, 25). Some protocols have added low doses of serum for promoting essential effects of activin A in the development of endodermal induction (26, 27). Furthermore, fibroblast growth factor (FGF) and Wnt signaling, which play important roles in normal liver development, are also effective in endodermal induction programs (28, 29). Researchers have also combined bone morphogenetic protein and FGFs to promote endodermal induction specificity (26, 30). Hepatocyte growth factor is also widely used in hepatic differentiation of pluripotent stem cell, mainly because of its ability in developing hepatoblast proliferation, migration, and survival through c-Met as tyrosine kinase part (31). Combination of FGF10 and retinoic acid with simultaneous inhibition of activin A is also another effective hepatic endodermal maturation protocol (32). In addition, oncostatin, which is a member of interleukin-6 family, in combination with glucocorticoids, can induce hepatocyte maturation (33, 34).

Genetic manipulation for the purpose of overexpression of specific genes involved in hepatic induction is another approach in regenerative medicine. Transducing some transcription factors such as Sox17, Gata, and hepatic nuclear factor 4α elevates iPSC hepatic induction at specific time intervals in culture media (35, 36). Furthermore, epigenetic interferences have also been used to improve hepatic differentiation protocols (32). For instance, sodium butyrate, a specific inhibitor of histone deacetylase, is frequently used to differentiate pluripotent stem cells into different cell lineages including hepatocytes in higher concentrations and longer time intervals (37–39).

Recent studies have proposed novel growth-factor-free protocols for the differentiation of pluripotent stem cells (40). Siller et al. (40) introduced a three-phasic protocol including 1) inhibition of glycogen synthase kinase 3 by CHIR99021 for definitive endoderm induction, 2) hepatic specification through dimethyl sulfoxide treatment, and 3) using dexamethasone and dihexa, a hepatocyte growth factor receptor agonist, to differentiate pluripotent stem cells into hepatocyte-like cells. Zhu et al. (41) also used a cocktail of small molecules for incompletely reprogrammed human fibroblast cells to hepatocytes. In addition, Shan. et al. (42) identified 12,480 small molecules in a liver platform, and they classified them into two large groups: functional proliferation hits and functional hits, which were able to promote the differentiation of iPSCs and the maturation of resulted hepatocyte-like cells. Improved directed redifferentiation using small molecules can improve results on a cost-benefit basis in large-scale applications.

Using viral vectors for transducing Oct4, Sox2, Klf4, and c-Myc is an exciting method for generating human iPSCs. Despite high efficacy of this procedure, there remain some critical limitations for the application of iPSCs in clinics (65). Applications of retroviral-generated iPSCs are limited because of the 1) integration of retroviral DNA into host genome with variable copy numbers which interfere with promoter elements, polyadenylation signals, and coding sequences, affecting transcription potency (20), and 2) loss of pluripotency potential because of low expression of exogenous Oct4, Sox2, Klf4, and c-Myc from viral constructs (66). Another critical concern is the exogenous genes itself; overexpression of Oct4, Sox2, Klf4, and c-Myc increases the chance of tumorigenesis (66, 67). Overexpression of Oct4 induces epithelial cell dysplasia (68). Sox2 overexpression is also associated with serrated adenoma and mucinous colon carcinoma (69). Klf4 and c-Myc are also associated with breast cancer and some other human carcinomas (70). Tumor progression is also observed in murine chimeras after injection of retroviral-generated iPSCs to blastocysts which has been attributed to c-Myc overexpression (71, 72).

Despite gained advances in the differentiation of pluripotent stem cells into functional hepatocytes in vitro, production of primary adult hepatocytes that can proliferate in vivo still remains inaccessible (73, 74). The reason for this problem is that expansion and proliferation of transplanted cells need a tense sustain of hepatic cell mass (75). Furthermore, besides pluripotent stem cells-related problems, cell delivery complications are another important limiting factor of liver cell therapy. Direct injection of cells to liver parenchyma may increase the risk of cell entry to hepatic vein outflow and pulmonary vein, causing embolic complications (76). Injection of cells to hepatic or splenic artery, theoretically, also seems to be achievable. However, these methods may increase the risk of tissue necrosis due to embolic occlusion of vessels. In high blood flow condition, engrafted cells may be destroyed because of incoming mechanical forces (47). Furthermore, in portal hypertension and chronic liver diseases, the transplanted cell may be translocated to lungs through portosystemic collaterals or channels causing cardiovascular problems (Figure 4) (45,77). Therefore, there is an urgent need for clinical trial designing for the application of successful cell delivery methods to liver sinusoids. Obviously, standards of professional practice play an important role in the clinical setting. There is no international standard for reprogrammed liver cell therapy, as cell therapy in general has been limited to heterologous primary cells with resource scarcity and little satisfactory outcome.

The major obstacle in hepatocyte transplantation is poor engraftment results, encouraging researchers to suggest new strategies. Prominent among these are modifying metabolic status in the recipients of liver cell therapy (78) and co-transplantation of mesenchymal stem cells (79) because of their significant effects on liver regeneration and repair.