Thinking outside the liver: Induced pluripotent stem cells for hepatic applications

Table 1 New approaches to reprogramming of differentiated cells to a pluripotent state

Method	Results of reprogramming	Drawbacks	Ref.
Transfer of the nucleus from a somatic cell to an enucleated oocyte	The somatic cell nucleus is reprogrammed in the oocyte, and a whole organism develops as a result. Patient-specific hESCs can be derived	Low efficiency. Developmental abnormalities in cloned animals. Ethical and legal restrictions	[24-28]
Fusion of ESCs with differentiated cells	Hybrids of differentiated cells and ESCs display all properties of pluripotent cells	Cell hybrids lack a normal diploid chromosome set	[29,30]
Reprogramming of somatic cells to a pluripotent state can be generated by the ectopic expression of 4 transcription factors, Oct4, Klf4, Sox2 and c-Myc	Somatic cells regain a pluripotent state and become similar in properties to ESCs	Low efficiency of iPSC derivation. Viral integration. Tumor formation	[7]

ESCs: Embryonic stem cells; hESCs: Human embryonic stem cells; iPSC: Induced pluripotent stem cell; Oct: Octamer-binding transcription factor; Sox2: SRY box-containing gene 2; Klf4: Kruppel-like factor 4.

Table 2 Various induction methods to generate induced pluripotent stem cells

Methods	Advantages	Disadvantages	Ref.
Retroviral vectors	High efficiency	Genome integration, dividing target cells needed	[7-9,32,41,42]
Lentiviral vectors	High efficiency, target cells need not be dividing	Genome integration	[47-49]
Lentiviral vectors with Cre/Lox	High efficiency	Minimize genomic integration	[43,44]
Piggyback transposon	Precise deletion is possible	Minimize genomic integration, laborious	[45,46]
Viral vectors	No genome integration	Low efficiency	[34-37]
Adenoviral vectors
Sendai vectors
DNA vectors
Plasmid vectors
Episomal vectors
Minicircle vectors
Protein transduction	No genome integration	Low efficiency	[38]
Small molecules	No genetic modification	Low efficiency	[39]
Synthetic mRNA	No genetic modification, high efficiency	Multiple rounds of transfection are needed	[40]

Table 3 Role of reprogramming factors for induced pluripotent stem cell generation

Reprogramming factors	Description	Function	Ref.
Oct4	Octamer binding transcription factor 4	This transcription factor plays a role in embryonic development, especially during early embryogenesis, and it is necessary for embryonic stem cell pluripotency	[7]
Sox2	SRY box 2	In embryonic stem cells, Sox2 and Oct3/4 often co-occupy target genes, including own promoters. These proteins cooperate regulatory feedback loops to maintain pluripotency	[60]
Klf4	Kruppel-like factor 4	This transcription factor plays a role in upregulation of pluripotency gene Nanog and the modification of chromatin structure to facilitate the binding of Oct3/4 and Sox2 to their sequences. Klf4 itself is an oncogenic factor. This gene is over expressed in a variety of tumor types associated with advanced cancer	[61-63]
c-Myc	Proto oncogene protein	An oncogene that induces global histone acetylation, allowing Oct3/4 and Sox2 to bind to their specific target loci	[60,63]
Nanog	Homeo box transcription factor	A transcription factor critically involved with self-renewal of undifferentiated embryonic stem cells	[64]
Lin28	RNA binding protein Lin28	The Lin28 gene codes for an RNA-binding protein that selectively blocks the processing of microRNAs of the let-7 family, and possibly certain other microRNAs in ESCs, to prevent their differentiation	[65,66]

ESCs: Embryonic stem cells; Oct: Octamer-binding transcription factor; Sox2: SRY box-containing gene 2; Klf4: Kruppel-like factor 4.

Table 4 Differentiation protocols for induced pluripotent stem cell-derived hepatocytes

Ref.	Species	Differentiation protocol	Remarks
Sullivan et al[78]	Human	Activin A, Wnt3a (3 d), Activin A (2 d), DMSO (3 d), HGF, OSM (6 d)	Generated functional hepatocyte-like cells from human-iPSCs
Song et al[79]	Human	Activin A (3 d), FGF4, BMP-2 (4 d), HGF, KGF (6 d), OSM, Dex (5 d) then OSM, Dex, N2B27 (3 d)	iPSCs had fewer expressed liver-enriched genes compared with human hepatocytes
Si-Tayeb et al[80]	Human	Activin A (5 d), bFGF, BMP-4 (5 d), HGF (5 d), OSM (5 d)	Transplanted hepatocyte-like cells into the lobe of newborn mice and demonstrated homing of donor cells
Liu et al[81]	Human	Activin A (5 d), FGF4, HGF (5 d ), Single Quotes (lonza), FGF4, HGF, OSM, Dex (10 d)	Human hepatocyte-derived iPSCs are able to differentiate into functional hepatocytes
Takata et al[82]	Human	Activin A ( 3 d), HGF (5 d), OSM (5 d)	Generated hepatocyte-like cells from iPSCs using three growth factors in a short time
Gai et al[83]	Mouse	Activin A, Wnt3 (6 d), bFGF, DMSO (3 d), HGF, DMSO (9 d), HGF, OSM, DMSO (7 d)	Generated hepatocytes from iPSCs
Iwamuro et al[84]	Mouse	Activin A, bFGF (3 d), HGF (5 d)	Generated hepatocyte-like cells from iPSCs

iPSCs: Induced pluripotent stem cells; DMSO: Dimethyl sulfoxide; HGF: Hepatocyte growth factor; OSM: Oncostatin M; Dex: Dexamethasone; FGF4: Fibroblast growth factor-4; BMP: Bone morphogenetic protein; KGF: Keratinocyte growth gactor; bFGF: Basic fibroblast growth factor.

Table 5 Direct conversion approaches for specific cell types

Ref.	Key factors	Direct converted cell type
Vierbuchen et al[109]	Brn2, Ascl1, and Myt1l	Transdifferentiated mouse fibroblasts into functional neuronal cells
Ieda et al[110]	Gata4, Mef2c, and Tbx5	Transdifferentiated mouse dermal fibroblasts into cardiomyocyte-like cells
Szabo et al[111]	Oct4	Transdifferentiated human fibroblast cells into hematopoietic progenitors
Huang et al[112]	Gata4, Hnf1α and Foxa3, and inactivation of p19Arf	Transdifferentiated mouse tail-tip fibroblasts into hepatocyte-like cell

Brn2: Brain-2; Ascl1: Achaete-scute homolog 1; Myt1l: Myelin transcription factor 1; Gata4: GATA binding protein 4; Mef2c: Myocyte enhancer factor 2; Tbx5: T-box protein 5; Hnf1α: Hepatocyte nuclear factor 1α; Foxa3: Forkhead box protein A3; p19: protein p19; Oct: Octamer-binding transcription factor.