Published online: 30 March 2010

# Springer Science+Business Media, LLC 2010

Abstract More recently, reprogramming of somatic cells to an embryonic stem cell-like state presents a milestone in the realm of stem cells, making it possible to derive all cell types from any patients bearing specific genetic mutations. With the development of induced pluripotent stem (iPS) cells, we are now able to use the derivatives of iPS cells to study the mechanisms of disease and to perform drug screening and toxicology testing. In addition, differentiated iPS cells are now close to be used in clinical practice. Here we review the progress of iPS technique and the possible application in the area of Parkinson’s disease treatment.

Keywords Parkinson’s Disease . iPS . Treatment .

Reprogramming

The Puzzle of Current Neurodegenerative Diseases

Treatment

Recently two long-term clinical evaluations of neural transplantation as potential treatment for neurodegenerative

Liang Xu and Yu-Yan Tan have contributed equally to this work

L. Xu : Y.-Y. Tan : J.-Q. Ding : S.-D. Chen (*) Department of Neurology and Institute of Neurology, Ruijin Hospital,

Shanghai Jiao Tong University School of Medicine, No.197, Rui Jin Er Road,

Shanghai 200025, China

e-mail: chen_sd@medmail.com.cn

L. Xu : Y.-Y. Tan : J.-Q. Ding : S.-D. Chen

Lab of Neurodegenerative Diseases, Institute of Health Science, Shanghai Institutes of Biological Sciences,

Chinese Academy of Science & Shanghai Jiao Tong University School of Medicine,

Shanghai 200025, China

diseases such as Parkinson’s disease [1] and Huntington’s disease [2] have been initiated in attempt to replace lost neurons and improve patient outcomes. In spite of marginal and transient clinical benefits demonstrated, some of the grafted cells seemed to have acquired signs of disease pathology. Alpha-synuclein and ubiquitin positive Lewy bodies were astonishingly found in implanted neurons in Parkinson’s disease trial, and the disease-like neuronal degeneration was observed in Huntington's disease trial. In addition, grafted neurons were intermingled with microglial cells, resembling an ongoing phagocytic event. These discouraging results raise uncertainty about this therapeutic approach for neurodegenerative diseases.

Introduction of iPS Cells

Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. In 2006, Japanese scientist Shinya Yamanaka and coworkers [3] first demonstrated the induction of pluripotent stem cells from adult mouse fibroblasts by introducing four factors, Oct4, Sox2, c-Myc and Klf4. In the following year, induced human pluripotent stem (hiPS) cells were successfully induced by Yamanaka and colleagues [4] using four factors Oct4, Sox2, c-Myc and Klf4 and by Thomson lab [5] using Oct4, Sox2, Nanog and Lin28. Despite disparity of transferring factors, these hiPS cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cellspecific genes, telomerase activity and potentiality to differentiate into advanced derivatives of all three primary germ layers. In 2009, Qi Zhou [6] confirmed that these induced embryonic-like pluripotent stem cells are capable

of generating viable, fertile live-born progeny by tetraploid complementation. The technology of such induced pluripotent human cells is a milestone not only in regenerative medicine but also in drug development, as well as for applications in transplantation medicine. Unlike somatic cell nuclear transfer (SCNT), iPS technique does not involve the use of embryos, so it is much less controversial and easier to perform.

Initially Yamanaka selected 24 genes as candidates to reprogram somatic cells to iPS cells and further restricted to four transcription factors Oct4, Sox2, Klf4, and c-Myc [3]. A little difference from Yamanaka, Junying Yu showed that Oct4, Sox2, Nanog and Lin28 are sufficient to reprogram human somatic cells to pluripotent stem cells [5]. So was four transcription factors method of direct reprogramming of human somatic cell to pluripotent stem cells established. As neural progenitor cells (NPCs) expressed high levels of endogenous Sox2, Eminli [7] found that viral Sox2 was not required in NPCs reprogramming process. Due to the random integration of multiple copies of the c-Myc oncogene into the target cell genome, researchers attempted to find the iPS induction safer and simpler [8]. Nakagawa [9] showed the generation of hiPS cells from adult dermal fibroblasts without c-Myc. Huangfu [10] utilized valproic acid (VPA)—a histone deacetylase inhibitor, to reprogram primary human fibroblasts with only two factors, Oct4 and Sox2, without the need for the oncogenes c-Myc or Klf4, further reduced the risk of tumorigenesis. In 2009, Kim reported that Oct4 alone is sufficient to directly reprogram adult mouse [11] or fetal human neural stem cells to iPS cells [12].

Many evidences have indicated that terminally differentiated cells can be reprogrammed to pluripotency. Aoi [13] successfully converted adult mouse hepatocytes and gastric epithelial cells to iPS cells. Stadtfeld [14] used inducible lentiviruses system to reverse pancreatic beta cells to iPS cells. A striking finding by Hanna [15] was that reprogramming mouse B lymphocytes to iPS cells with four transcript factors combined with either ectopic expression of the transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBP alpha) or specific knockdown of the B cell transcription factor Pax5. These results provide genetic proof that terminally differentiated cells can be reprogrammed into pluripotent cells regardless of cell types or differentiation stages.

The time needed for reprogramming somatic cell to pluripotent stem cells differed from one lab to another. The classical method from Yamanaka indicated that 16 days of four factors transduction have been sufficient to form G418-resistant colonies [3]. Mali [16] added SV40 large T antigen during the reprogramming course to enhance the efficiency by 23–70-fold from both human adult and fetal fibroblasts and shorten the time by 1-2 weeks. Maherali

[17] created a doxycycline-inducible lentiviral system, which converted primary human fibroblasts and keratinocytes into hiPSCs in 10 days, and obtained “secondary” hiPSCs at a frequency at least 100-fold greater than the initial conversion. Hockemeyer [18] adopted the same idea and achieved as high as up to 2% of the reprogrammed pluripotent “secondary” human iPSCs. Huangfu [19] used VPA to improve reprogramming efficiency by more than 100-fold. Ying Jin’s lab reported [20] the most rapid period of 6 days after viral infection from human amniotic fluidderived cells (hAFDCs) to iPS cells.

Most instances of direct reprogramming have been achieved by viral vectors in which both the vector backbone and transgenes are permanently integrated into the genome, raising the risk of tumorigenicity and unpredictable genetic dysfunction. Ectopic expression of either Oct4 or Klf4 can induce dysplasia [21], and c-Myc causes tumorigenicity in offspring [22], so scientists attempt to search for nonintegrated and safer methods to reprogram human somatic cell to pluripotent stem cell. Stadtfeld [23] adopted adenoviruses transiently expressing Oct4, Sox2, Klf4, and c–Myc to generate mouse iPS cells. Okita [24] utilized repeated transfection of two expression plasmids to achieve iPS cells without evidence of plasmid integration. However, the efficiency of both approaches is extremely low. In 2009, Soldner [25] described efficient reprogramming of fibroblasts from five patients with idiopathic Parkinson’s disease to iPS cells. The shining point was that the transgene can be removed once reprogramming has been achieved by Cre/LoxP recombination. Based on the similar idea of Cre/LoxP system, Kaji [21] adopted a single multiprotein expression vector comprising of the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides. This approach successfully excised integrated transgenes, but still left some of vector residuals which were high risky for gene mutation, so the complete virusfree induction of pluripotency needs to be developed to solve the safety issue of the reprogramming. One possible way to avoid genetic modification of targeting cell genome is to directly deliver reprogramming protein into cells. Zhou et al. [26] and Kim et al. [27] designed and fused a polyarginine (11R or 9R) protein transduction domain to the four reprogramming factors and generated mouse or human iPS cells respectively, though the reprogramming from fibroblast to iPS cell requires multiple protein treatment circles and double time of that of viral iPS transduction. Different from direct delivery of reprogramming proteins, Yu [28] reported a strategy that a single transfection with oriP/EBNA1 (Epstein-Barr nuclear antigen-1)–based episomal vectors was applied to produce hiPS cells from fibroblasts completely free of vector and transgene sequences. The oriP/EBNA1 vectors would be lost (5% per cell generation) with the drug selection withdrawal, so the cell

devoid of episomes could be easily isolated. In Woltjen’s method [29], piggyBac (PB) transposition/transposase system was introduced to iPS production. This system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. By taking advantage of the PB system, the author showed the individual PB insertions providing a marker for discovery and a traceless removal of PB transposition. The strategies of nonintegrated induction of iPS provide strong tools for future cell-based therapy. Currently the methods of nontransgene integration induction of iPS cells are successfully established in murine and human cells, albeit low reprogramming efficacy (Table 1). Among the strategies, the combination of protein induction and small molecules to reprogram human cells are of most interests and expected to apply to clinical use. A simple, standardized and reproducible reprogramming protocol is the first and important step.

The hiPS cells are usually established and maintained on mouse embryonic fibroblast feeder (MEF) layers, but the xenosupport systems will increase the risk of animal pathogens transmission from animal to iPS cells. Currently many human feeder cells have been discovered to maintain the undifferentiated state of human stem cell, including human uterine endometrial cells [30], human fibroblasts [31, 32], and human adult marrow cells [33]. Recent study of Yamanaka has demonstrated that hiPS cells can be established and maintained on isogenic parental feeder cells [34], moving forward to clinical application. In addition, feeder free culture [35] and defined feeder-independent medium [36] are developed to make the culture of human stem cell consistent and reliable. A study [37] was performed last year to compare current serum-free and defined culture media commercially available for human embryonic stem cells lines. For defined StemPro and mTeSR1 media, the cells were cultured and maintained feeder-free on culture dishes coated with extracellular matrices, with no requirement of feeder conditioned media. For xeno-free HESGRO and KnockOut media (XSR), mitotically-inactivated human foreskin feeders were required. The xeno-free and defined culture medium for human stem cell maintenance and passage needs to be combined and improved to move forward to clinical use.

iPS and Parkinson’s Disease

When a series of human healthy somatic cells are reprogrammed to iPS cells, scientists highlight on the pathologic cells from patients, especially the patients suffering genetic diseases or genetics associated diseases, expecting that reversed genetically deficient iPS cells can be modified and corrected so as to make the treatment back to the same

patient where iPS cells are from. This idea really worked in the first step. Dimos’ [38] and Park’s [39] experiments supported the assumption that iPS cells could be generated from patients with a variety of genetic diseases, including amyotrophic lateral sclerosis (ALS), adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21, and the carrier state of Lesch-Nyhan syndrome. Such disease-specific stem cells provide an invaluable resource for medical research and drug development.

Parkinson’s disease is a common progressive bradykinetic disorder characterised by the presence of severe parscompacta nigral neuronal loss, and accumulation of aggregated alpha-synuclein. Genetic studies have shown that several mutations in several genes are linked with L-dopa- responsive Parkinsonism. These genes include LRRK-2, parkin, DJ-1, PINK1, ATP13A2, alpha-synuclein and GBA. Dopamine replacement therapy considerably reduces motor handicap, and effective treatment of associated depression, pain, constipation, and nocturnal difficulties can improve quality of life [40]. Stem cells and gene therapy are promising research therapeutic approaches.

Currently there are four strategies to induce reprogramming: cell fusion, cell culture, nuclear transfer and direct reprogramming [41, 42]. Apart from cell fusion which produces tetraploid cells, all of the rest are applicable to disease treatment.

The ES cell based therapy is complicated by immune rejection due to immunological incompatibility between patient and donor ES cells. Furthermore, recently two reports [43, 44] of long-term trials illustrated alpha-synuclein- positive Lewy bodies in grafted neurons, suggesting the spreading of Parkinson’s disease to some of the transplanted cells in a manner similar to host dopaminergic neurons in the substantia nigra. The mechanism underlying the unexpected spreading of alpha-synuclein pathology of the grafted dopaminergic neurons in a similar manner of pathological changes in the Parkinson’s disease are obscure. The unfavorable microenvironment may be responsible for this. Evidences from survey of Parkinson’s disease patients and animal experiments have shown that drugs [45, 46] and toxins [47] can result in hostile microenvironment to the transplanted dopaminergic neurons. Microglial activation and impaired neurotrophic factors in Parkinson’s disease could also contribute to alpha-synuclein aggregation in grafted cells [48]. The hypothesis of “permissive templating” [49] indicates that the misfolded alpha-synuclein leads to aggregation and deposition in the host, and consequently transmits into grafted cells in a “prion-like” mechanism. Of note, the abundance of alpha-synuclein and the formation of Lewy

bodies could not be detrimental to grafted dopaminergic neurons, and the duration of efficacy of cell replacement therapy may not be dramatically limited. The findings remind us that stem cell replacement therapies for Parkinson’s disease may not be as good as we had hoped for. More mechanism needs to be explored to understand Parkinson’s disease.

The successful generation of cloned stem cells and animals by SCNT created the possibility to generate genetically identical SCNT-ES cells by using donor cells from a patient [50], but both technical and ethical

considerations of the nuclear transfer procedure impede the practical realization of “therapeutic SCNT” in human.

Once the iPS cells are on hand, the next step is to develop Parkinson’s disease specific stem cells and differentiate them to defined dopaminergic neurons. Right now Parkinson’s disease specific stem cells have been induced [25, 39]. The reprogrammed iPS can be differentiated into dopaminergic neurons [25]. The protocol available for stem cell differentiation is based on human embryonic stem cells. The classic differentiation from stem cell to dopaminergic

neuron was established by McKay consisting of five stages: expanding undifferentiated ES cells, generating embryoid bodies, selecting and expanding nestin positive cells, and inducing differentiation [51]. In 2008, Cho et al. [52, 53] developed a protocol, yielding a large-scale of functional dopaminergic neurons from human embryonic stem cells with high efficiency. The unique feature of this method is the generation of homogeneous spherical neural masses (SNMs) from the human ES cell-derived neural precursors. As a result, SNMs can be coaxed into dopaminergic neurons as high efficiency as 86% tyrosine hydroxylasepositive neurons/total neurons and it takes only 14 days.

Whether the converted iPS cells could be therapeutic for the patients of Parkinson’s disease are still uncertain, but two examples raised the hope of utilization of this potential tool. One is from Hanna’s study which rescued humanized sickle cell anemia mouse by correction of the human sickle hemoglobin allele by gene specific targeting after transplantation with hematopoietic progenitors obtained in vitro from autologous iPS cells. iPS cells were used to treat sickle cell anemia in mice, demonstrating that iPS cells had real therapeutic potential [54]. The other instance is so stimulating that Wernig and colleagues used programmed pluripotent stem cells to improve the symptoms of Parkinson’s disease in mice [55]. The NPCs differentiated from iPS cells, upon transplantation into the fetal mouse brain, migrated into various brain regions, differentiated into glia and neurons including dopaminergic neurons, and improved behavior in a rat model of Parkinson’s disease. This is the first study to specifically use reprogrammed cells to combat Parkinson’s disease.

The generation of Parkinson’s disease specific iPS cells dispels the fear of immune rejection in allogeneic or xenogeneic transplantation in cell therapy and resolves the problem of shortage of large scale of transplanted cells. As many patients suffering Parkinson’s disease are associated with carrying of the susceptible genes, the occurrence of iPS technology provides the opportunity of genome modification to rectify the mutant gene in the level of initial stem cells and of differentiating them to dopaminergic neurons for cell replacement. Moreover, the comparison of cells between healthy individuals and Parkinson’s disease patients or from different developmental stage helps to screen for the discrepancy of gene expression, find the pivotal proteins of etiopathogenesis, thereby elucidate the pathogenesis of Parkinson’s disease.

Summary

Reprogramming of the human somatic epigenome to a pluripotent, embryonic state through ectopic expression of the defined transcription factors is a breakthrough in

biological science. It thus solves the problem of ethical considerations and immune rejection, offering an unprecedented opportunity to regenerative medicine, drug screening and full exploration of the reprogramming process and future cell-based therapies. Current reprogramming strategies differ from one lab to another in cell resource, factor delivery, culture condition and iPS identification. A standardization of certain parameters of the reprogramming process needs to be formulated to compare the present results across the diverse labs and exploit the abundance of new information in future [56, 57]. Besides, effective restricted differentiation to dopaminergic neurons to replace the cell loss in the substantia nigra is essential for the treatment of Parkinson’s diseases. Many obstacles are still to be overcome to fully understand and effectively apply this powerful technique.

Acknowledgements This work was supported by grants from the National Program of Basic Research (2006CB500706, 2007CB947900,2010CB945200) of China, National Natural Science Fund (30872729, 30971031), Shanghai Key Discipline Program (S30202), and Program for Outstanding Medical Academic Leader (LJ 06003).

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