fibroblast-like cells derived from ips cells of patients...

Journal of Stem Cell Research & Therapeutics

Fibroblast-Like Cells Derived from iPS Cells of Patients with the Familial forms of Parkinson’s Disease can Serve an Effective Feeder for Derivation and Cultivation of New

iPS Cells Lines

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IntroductionGeneration of induced pluripotent stem cells (iPSC) is

considered one of the major biological discoveries of the 21st century. IPSC were originally obtained in 2006-2007 by Japanese scientists first from mouse fibroblasts, and later from adult human fibroblasts [1,2]. IPSC have provided a unique platform for studying pathogenesis of different diseases and opened the door to their clinical application in regenerative medicine [2]. The employment of iPSC does not entail ethical issues, since adult human tissues are used just as a source for their generation. Regenerative medicine has great expectations for that particular cell type, since immune rejection represents one of the main complications in tissue and organ grafting.

All patients after surgery are doomed to a long-term or even lifelong immunosuppressive therapy. This leads to high susceptibility of humans to various infections, and to the development of complicated diseases of liver, kidney and pancreas, and furthermore, the donor organ can still be rejected. Patient-specificity of iPSC puts them on a completely different level, as their differentiated progeny is autologous to the patient, and will not be rejected, since these iPSC contain an identical set of antigens. The generation of new iPSC lines and their further propagation is typically performed on a feeding layer (feeder) of

mouse embryonic fibroblasts (MEF), human fibroblasts derived from newborn foreskin, specially designed commercial substrates Matrigel, Laminin 521 matrix, CELL start, and on some recombinant proteins [3-7]. However, in the case of regenerative medicine the utilization of cells of animal origin during manipulations with iPSC should be completely avoided. Such a co-culture of cell lines can lead to the transmission of known and obscure xenogenic infectious pathogens. A group of scientists from the University of California, San Diego, and from the Salk institute have detected expression of the xenogenic factor Neu5Gc (N-Glycolylneuraminic acid) on the surface of human embryonic stem cells (hESC) after long-term co-culturing with murine fibroblasts, thus confirming the possibility of such a transmission [8]. Any foreign agents in cell cultures intended for transplantation will induce negative immunological reactions in the recipient, and consequently decrease the therapeutic effects.

An alternative method for iPSC derivation and culture is the use of commercial substrates. However, for this method, a number of authors reported a decreased efficiency of reprogramming and a decreased quality of the cellular material [4,9-11]. Thus, a prolonged cultivation of hESC on a Matrigel substrate was reported to induce genomic instability affecting chromosomes 12 and 17 [12]. To date, it can be concluded that none of the above methods for iPSC generation and culture can be considered

Volume 3 Issue 3 - 2017

1Department of Viral and Cellular Molecular Genetics, Institute of Molecular Genetics, Russia2Department of Brain Research, Research Center of Neurology, Russia

*Corresponding author: Igor A Grivennikov, Department of Viral and Cellular Molecular Genetics, Institute of Molecular Genetics, Russian Academy of Sciences, 123182 Moscow, Kurchatov sq.2, Russian Federation, Russia, Tel: +7(499)-1960014; Fax: +7(499)-1960221; Email: ; Received: September 10, 2017 | Published: October 16, 2017

Research Article

J Stem Cell Res Ther 2017, 3(3): 00102

Abstract

Induced pluripotent stem cells (iPSC) can serve a highly informative model of hereditary and sporadic forms of human diseases. In prospect, such cells might be used in substitutive and regenerative therapy of a wide spectrum of diseases, for treatment of traumas and thermal injuries. At present, mouse embryonic fibroblasts are frequently used for obtaining and culturing human ES and iPS cells, whereas a real risk of transmission of xenogenic infectious pathogens makes it impossible to use these cells in regenerative medicine. It was shown that iPSC obtained from patients with familial forms (mutations in PARK2 and PARK8 genes) of Parkinson’s disease can effectively differentiate into fibroblast-like cells (derivatives), similar to iPSC generated from healthy donors. It was demonstrated that the obtained derivatives can be effectively used as feeder layers not only to maintain the pluripotency of autologous and allogeneic iPSC, but also to obtain new patient specific iPSC lines.

Keywords: Induced pluripotent stem cells; Parkinson’s disease; Derivatives; Reprogramming; Proliferation; Fibroblast-like cells; Feeder layer

Abbreviations: iPSC: Induced Pluripotent Stem Cells; HESC: Human Embryonic Stem Cells; MEF: Mouse Embryonic Fibroblasts; Neu5Gc: N-Glycolylneuraminic Acid; PD: Parkinson’s Disease; EBs: Embryoid Bodies

Citation: Novosadova EV, Manuilova ES, Arsenyeva EL, Tarantul VZ, Illarioshkin SN et al. (2017) Fibroblast-Like Cells Derived from iPS Cells of Patients with the Familial forms of Parkinson’s Disease can Serve an Effective Feeder for Derivation and Cultivation of New iPS Cells Lines. J Stem Cell Res Ther 3(3): 00102. DOI: 10.15406/jsrt.2017.03.00102

Fibroblast-Like Cells Derived from iPS Cells of Patients with the Familial forms of Parkinson’s Disease can Serve an Effective Feeder for Derivation and Cultivation of New iPS Cells Lines

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absolutely safe. In this regard, it is extremely important to find a substrate that meets all the requirements for the “purity” of iPSC. One of the possible solutions to this problem is to use an autologous or allogenic feeder derived from iPSC and ESC themselves. The first attempts to produce this type of feeder were performed in 2004 by Hu et al. [13]. The scientists have performed a directed differentiation of hESC of H1 line into fibroblast-like cells (derivatives) that were subsequently immortalized by introducing the pBABE-hTERT plasmid (carrying the telomerase reverse transcriptase gene). The derivatives and their conditioned media were shown to be capable to maintain the pluripotent state of ES cells [13]. Further, several research groups have presented data indicating the possibility of using autologous derivatives to maintain the undifferentiated state of hESC [14-16]. Derivatives generated from hESC and iPSC possess the attributes of stromal fibroblasts [17] and demonstrate a comparable level of expression of extracellular matrix proteins, while their levels of expression of type I and V collagens and fibronectin are significantly higher than those in newborn foreskin-derived fibroblasts [18]. It is important to note that derivatives can be used not only for propagation of pluripotent cells, but also for generation of new iPSC and ESC lines [19,20]. Recently we supported these data using fibroblast-like cells obtained from iPSC of healthy donors [21]. A detailed study of molecular and functional properties of derivatives was performed using iPSC from healthy donors. At the same time, the data on the generation of iPSC derivatives bearing mutations in the genes involved in the development of human neurodegenerative diseases are lacking in the literature.

In this study, we demonstrated the possibility to generate fibroblast-like derivatives from iPSC from patients with familial forms of Parkinson’s disease (mutations in PARK2 and PARK8 genes) and showed their ability to serve as an effective feeder layer for cultivation and generation of the new patient specific iPSC lines.

Materials and Methods

iPSC cultures

The following human cell cultures were used: fibroblasts obtained from the skin of patients with Parkinson’s disease and from a healthy donor, were kindly provided by SN Illarioshkin (Scientific Center of Neurology, Moscow, Russia); iPSC lines from a healthy donor (IPSRG2L) and from patients with familial forms of Parkinson’s disease (PD) IPSPDP1.5L and IPSPDL2.15L, were kindly provided by SL Kiselev & MA Lagarkova [22] (Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russia). IPSC were cultured under the standart condition [22].

Generation of embryoid bodies by the “hanging drop” method

IPSC colonies were detached with Dispase (Gibco, USA). To this end, the culture medium was removed, the cells were washed with DMEM medium (Paneco, Russian Federation), treated with 1 ml of Dispase solution (1 mg/mL) per 35 mm dish, and incubated for 7-10 min at 37°С. The enzyme solution was then discarded, cells were washed 5 times with 1 ml of DMEM medium, and then 1 ml of mTeSr medium was added. The colonies were detached with a 200 μl tip, carefully resuspended into a single cell suspension,

and the cells were counted in a hemocytometer. Several 20 μl drops containing 1000-4000 cells were transferred onto the inner surface of 60 mm Petri dish lids. To prevent medium evaporation, the dish was filled with 5 ml of DMEM medium, covered with a lid with the drops and placed into a CO2 incubator at 5% CO2, 37°C.

Generation of derivatives

On the second or third day of incubation, the embryoid bodies (EBs) formed in drops were transferred onto a 24-well plate (coated with 0.1% gelatin solution or Matrigel), one EB per well with EB growth medium: DMEM/F12 (Gibco, USA), 20% fetal bovine serum (FBS) (Hyclone, USA), 2 mM L-glutamine (ICN Biomedicals, USA), 0.1 mM β-mercaptoethanol (Sigma, USA), 1% mixture of non-essential amino acids (Paneco, Russian Federation). The medium was changed every 48 h. When the cells reached monolayer confluency, they were dissociated with 0.25% trypsin solution, the EB debris were mechanically removed, and the cells were replated from each well onto 35 mm Petri dishes with EB medium. After 24 h, the medium was changed to: DMEM, 15% fetal bovine serum (Hyclone, USA), 2 mM L-glutamine (ICN Biomedicals, USA), 1% mixture of non-essential amino acids (Paneco, Russian Federation), penicillin- streptomycin (50 U/ml; 50 mg/mL) (Paneco, Russian Federation).

Cryopreservation of derivatives

Derivatives were frozen at the logarithmic phase of growth, at 500,000 cells per 1 ml of cryopreservation medium. Cells were dissociated with 0.25% trypsin solution and centrifuged at 350 x g. The supernatants were discarded, and the cells were gently resuspended in cryopreservation medium containing 90% FBS (Hyclone, USA), and 10% DMSO (Applichem Pancreas, Spain). The cells were then stored in liquid nitrogen.

iPSC cultures on the feeder cell layer

The medium for culturing human iPSC on derivatives had the following composition: DMEM-F12 (Gibco, USA), 20% serum replacement (SR) (Gibco, USA), 2 mM L-glutamine (ICN Biomedicals, USA), 0.1 mM β-mercaptoethanol (Sigma, USA), 1% non-essential amino acid mix (Paneco, Russian Federation), recombinant bFGF (4 ng/ml) (Peprоtech, USA), penicillin-streptomycin (50 U/ml; 50 mg/mL) (Paneco, Russian Federation).

To prevent proliferation of the feeder cells, Mitomycin C (ICN Biomedicals, USA) was added to culture medium at a final concentration of 3μg/ml, and the cells were incubated for 2 h at 37°C and 5% CO2, washed three times with DMEM, and then cultured in growth medium. IPSC were cultured at 37°С and 5% CO2 on a feeder layer of derivatives inactivated with mitomycin C in gelatinized 35 and 60 mm Petri dishes (Greiner, USA). Prior to plating of iPSC, derivative culture medium was replaced with iPSC culture medium in dishes with fresh feeder layers. Cells were passaged every 5-6 days by mechanical dissociation of colonies or by their digestion with dispase.

Polymerase chain reaction coupled with reverse transcription

Total RNA was extracted using a Ribozol A kit (AmpliSens Biotechnologies, Russian Federation) according to the manufacturer’s protocol. The resulting RNA was treated with

http://dx.doi.org/10.15406/jsrt.2017.03.00102



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DNase I (Fermentas, USA) and then subjected to reverse transcription procedure using M-MLV reverse transcriptase (Sileks, Russian Federation), according to the manufacturer’s

protocol. cDNA was then used for PCR-amplification with Taq polymerase (Sileks, Russian Federation). The primers used in PCR assay are presented in Table 1.

Table1. Primers used for PCR assay.

Gene Name Primer Sequence Annealing Т0С Amplicon Size (bp)

4-Oct 5’-CGACCATCTGCCGCTTTGAG-3’5’-CCCCCTGTCCCCCATTCCTA-3’ 69 572

sox 2 5’-TCCTGATTCCAGTTTGCCTC-3’5’-GCTTAGCCTCGTCGATGAAC-3’ 69 479

Nanog 5’-CAGCCCTGATTCTTCCACCAGTCCC-3’5’-TGGAAGGTTCCCAGTCGGGTTCACC-3’ 69 390

Foxd 3 5’-CAAGCCCAAGAACAGCCTAGTGAA-3’5’-TGACGAAGCAGTCGTTGAGTGAGA-3’ 57 202

Hesx 1 5’-ACCTGCAGCTCATCAGGGAAAGAT-3’5’-AAAGCAGTTCTTGGTCTCGGCCT-3’ 66 202

klf 4 5’-GCGCTGCTCCCATCTTTCT-3’5’-GGGGGGAAGTCGCTTCATGT-3’ 69 124

Gapdh 5’-GAAGGTGAAGGTCGGAGTCA-3’5’-TTCACACCCATGACGAACAT-3’ 60 401

Col I 5’-CCTCCTGACGCACGGCCAAG-3’5’-CCCTCGACGCCGGTGGTTTC-3’ 60 246

Col III 5’-CCTCCAACTGCTCCTACTCG-3’5’-TCGAAGCCTCTGTGTCCTTT-3’ 60 497

Col IV 5’-CCAGGATTTCAAGGTCCAAA-3’5’-TCATTGCCTTGCACGTAGAG-3’ 60 464

Col V 5’-CTGGGGAGAAGGGAAAACTC-3’5’-TCAGTCCAAGAGCTCCCACT-3’ 60 600

Vimentin 5’-ATTCCACTTTGCGTTCAAGG-3’5’-CTTCAGAGAGAGGAAGCCGA-3’ 60 96

Fibronectin 5’-CCATCGCAAACCGCTGCCAT-3’5’-AACACTTCTCAGCTATGGGCTT-3’ 60 112

Spontaneous differentiation of iPSC into derivatives of the three germ layers

Spontaneous differentiation of IPSC into derivatives of the three germ layers was performed according to the method described in [23].

Immunofluorescent staining of cell cultures

Immunofluorescent staining of cell cultures was performed according to the method described in [23].

Generation of new iPSC lines

Reprogramming of dermal fibroblasts was performed using a CytoTune®-iPS 2.0 Sendai Reprogramming Kit, (Invitrogen, USA) in accordance to the manufacturer’s recommendations.

On day 7 of culturing, the transfected fibroblasts were transferred onto mouse embryonic fibroblasts (MEF), autologous fibroblasts and derivatives inactivated with mitomycin C. For this purpose, MEF, autologous fibroblasts and derivatives were plated onto 100 mm Petri dishes (coated with 0.1% gelatin) at 650 000 cells per dish containing 10 ml of fibroblast growth medium. The next day, the cells were treated with mitomycin C and covered with fibroblast growth medium. In 24 h, these substrates were used for seeding transfected fibroblasts at 50 000, 100 000 and 150 000 cells per dish in fibroblast growth medium. The next day, growth medium was replaced with iPSC medium, consisting of DMEM/F12 (Gibco, USA), 20% serum replacement (Gibco, USA), 1 mM non-essential amino acids (Paneco, Russian Federation), 2 mM L-glutamine (ICN Biomedicals, USA), 55 μM β mercaptoethanol (Sigma, USA), 50 U/ml penicillin/streptomycin




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(Paneco, Russian Federation), and 4 ng/mL recombinant β-FGF (Peprоtech, USA). On day 25-27 of cultivation, the iPSC colonies were transferred onto a Matrigel substrate in mTeSR medium. The derived iPSC lines were then propagated, analyzed and subjected to cryopreservation.

Results

Generation of iPSC derivatives from healthy donors and patients with familial forms of Parkinson’s disease

In the current study, we used iPSC lines previously obtained from a neurologically healthy donor IPSRG2L (line iPSC N ) and from patients with familial forms of PD: IPSPDP1.5L (line iPSC PARK) derived from a patient bearing a mutation in the Parkin 2 gene (202-203 AG deletion in the 2nd exon) plus a splicing mutation in the first intron (IVS1+1G/A)), and IPSPDL2.15L (line iPSC LRRK) derived from a patient with a mixed form of PD: with a heterozygous mutation in leucine-rich repeat kinase 2 (LRRK2) at exon 41 (locus PARK8, G2019S), and a major mutation in the glucocerebrosidase (GBA) gene at exon 9 (N370S). All the cell lines used in this study had the normal diploid chromosome set and expressed a classic spectrum of pluripotency genes [24,25]. We have generated embryoid bodies from three iPSC lines (N, LRRK and PARK) by using the “hanging-drop” method.

2-3 day EBs were transferred onto a 24 well plate with EB growth medium, at one body per well. The plates were pre-treated with either 0.1% gelatin or Matrigel. Since EBs equally well attached to both substrates, in the following experiments we used only gelatin. In 36 h, the growth of fibroblast like cells (derivatives) could be observed around the attached EBs (Figure 1).

On day 5-6 after plating, the derivatives reached monolayer confluency and were passaged 1:2 on new Petri dishes. The EB

debris seen as dense clusters of cells (as in the center of Figure 1) were mechanically removed during passaging that allowed to obtain a homogeneous population of derivatives without visible contamination with other cell types.

Thus, we obtained three independent lines of derivatives: der N from healthy donor iPSC (iPSC line N), der LRRK from iPSC of a patient bearing mutations in the LRRK2 and GBA genes (iPSC line LRRK), and der PARK from iPSC of a patient with a Parkin gene mutation (iPSC line PARK) (Figure 2). After the first passage, the derivatives were cultured in EB growth medium which was subsequently replaced with a poorer medium used for routine culturing of fibroblasts (see Materials and Methods).

Figure 1: Derivatives generated in 36 h. after replating of EBs formed from 2 000 iPSC. A – fibroblast-like cells (derivatives), B-EB debris (х100).

Figure 2: Monogenic culture of derivatives, second passage (x100).

Characterization of the obtained derivatives

Expression of extracellular matrix (EM) proteins such as fibronectin, elastin, and type I, III, IV and V collagens is an immunophenotypic feature of fibroblasts. All the generated

derivatives were assayed for expression of fibroblast-specific genes using RT-PCR and immunocytochemistry. As a result, we demonstrated the expression of mRNA for type I, III, IV and V collagens, vimentin and fibronectin in all investigated cell lines (Figure 3).




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Using immunocytochemistry, we also revealed the presence of type I, III, IV collagens and fibronectin in all examined cell lines.

As a positive control, we used fibroblasts obtained from a healthy donor and previously used for generation of iPSC line N (Figure 4).

Using the obtained derivatives as a feeder for cultivation of different iPSC lines

It was previously demonstrated that fibroblast-like cells derived from hESC and iPSC from healthy donor are capable of supporting growth and maintaining the pluripotency of hESC and IPSC [18-20]. In our study, we have found that the derivatives obtained from hESC line H9 can maintain the pluripotency of iPSC from a healthy donor (line iPSC N) and from PD patients (lines iPSC LRRK and iPSC PARK) (unpublished data). Here, we first demonstrated generation of derivatives from iPSC from patients with different familial forms of PD. All the derivatives demonstrated a morphological and genetic similarity with primary fibroblasts. The effectiveness of generation of derivatives, as well as their proliferative activity, was equal for iPSC from PD patients and from a healthy donor. We investigated the ability of the obtained derivatives der N, der LRRK and der PARK to support the growth and maintain the pluripotency of both autologous and allogeneic iPSC (Figure 5). All iPSC were cultured for at least 6 passages on the derivatives and then assayed by RT-PCR for expression of genes responsible for pluripotency (Figure 6).

Figure 3: Expression of fibroblast-specific genes in the derivatives obtained: 1- der N, 2- der LRRK, 3-der PARK, 4-primary fibroblasts from a healthy donor.




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Figure 4: Immunofluorescent staining with antibodies to collagen type I - A, collagen type III - B, collagen type IV – C, and fibronectin - D. Nuclei were counterstained with DAPI (blue). (x 100).

Further, these iPSC lines were transferred to a feeder-free culture system (using Matrigel) and then propagated for 5 more passages. Under these conditions, all iPSC retained undifferentiated cell morphology. To analyze expression of markers specific to pluripotent cells, we performed an immunocytochemical staining with antibodies to SSEA-4 and Sox2, which identified these specific antigens in all analyzed iPSC lines (Figure 7).

Using the obtained derivatives as a feeder during generation of new iPSC lines

At the next stage, the derivatives of der LRRK line, bearing a double mutation in the LRRK2 and GBA genes, were used as a

Figure 5: Scheme of plating different iPSC lines onto the obtained derivatives.

Figure 6: RT-PCR analysis for the expression of the genes responsible for pluripotency. 1, der N / iPSC N; 2, der N / iPSC LRRK; 3, der N / iPSC PARK; 4, der LRRK / iPSC N; 5, der LRRK / iPSC LRRK; 6, der LRRK / iPSC PARK; 7, der PARK / iPSC N; 8, der PARK / iPSC PARK; 9, der PARK / iPSC LRRK.




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feeder layer to produce new iPSC lines from a 60 year old patient suffering with PD caused by a mutation in the GBA gene. As a control, we used autologous primary fibroblasts and MEF. Dermal fibroblasts of the patient were transfected with Sendai virus carrying the Oct3/4, Sox2, Klf4 and c-Myc transcription factors. According to the protocol, on day 7 after transfection, the cells were transferred onto Petri dishes in which der LRRK derivatives, MEF or autologous primary fibroblasts were used as a feeder layer. On day 5 of culturing, we observed a marked increase in growth of cell colonies on all substrates (Figure 8). However, starting from day 7, the colonies plated on an autologous feeder started to lose their structure and died on day 14-16.

On day 25-27 of culturing, individual colonies from both substrates (MEF and derivatives) were transferred onto new Matrigel-treated Petri dishes with mTeSR iPSC propagation medium.

The iPSC lines GBA/derivatives (obtained on derivatives) and GBA/MEF (obtained on MEF) were assayed for the expression of the pluripotency marker and for their ability to generate derivatives of the three germ layers. These results indicated that iPSC obtained both on derivatives and MEF possessed a pluripotent phenotype, and that they were able to form embryoid bodies and differentiate into mesodermal, ectodermal and endodermal lineages (Figure 9).

DiscussionAs mentioned above, iPSC are generated using feeder cell

layers of animal or human origin, or using specially designed

Figure 7: Expression of pluripotency markers Sox2 and SSEA4 in iPSC after 6 passages on derivatives. (x 100).

Figure 8: iPSC colonies on feeders. a, d – derivatives, b, e – MEF, c, f – autologous feeder (primary fibroblasts).

a,b,c- 5 days of cultivation, d,e,f- 12 days of cultivation (х 100).

Figure 9: Immunofluorescent staining of iPSC lines GBA/derivatives and GBA/MEF.

A - pluripotency markers SEEA4 (green), Oct4 (red), dapi (blue); B- endodermal marker alpha-fetoprotein (red), mesodermal marker - desmin (green), DAPI (blue); C - ectoderm marker - β-III tubulin (green), Sox1 (red), dapi (blue). (x 200).




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substrates [3-7]. Each technique has its pros and cons. Use of murine fibroblasts can lead to xenogenic infection, while human allogenic cells may be a source of viral, bacterial, parasitic and fungal infections. Commercial substrates usually consist of products of animal origin, for example, the commonly used Matrigel includes growth factors, type IV collagen and laminin and represents a product obtained from a soluble basal membrane extract of murine Engel berth-Holm-Swarm sarcoma cell line. Thus, there is a small but real risk of transmission of xenogenic material. A possible solution to this problem is autologous feeder cells derived from the unique patient. Methods for obtaining skin explants and propagation of biomaterial from them are well established and possess no technical difficulties. However, not all generated fibroblast lines are able to provide the factors necessary for iPSC and to maintain them in a pluripotent state. Takahashi et al. [20] have tested 14 dermal fibroblast lines and demonstrated that three of them were unable to maintain iPSC in an undifferentiated state after the second passage. All these fibroblast lines were obtained from aged donors (68-, 77- and 81-year old) [20]. However, the authors themselves are not inclined to consider the donor age as the sole reason for this failure, since two other cell lines generated from 69- and 73-year old donors successfully maintained the pluripotency of iPSC. The same team of researchers was able to obtain new iPSC lines using dermal fibroblasts from 26-, 56- and 73-year old donors [20].

In the current work, we used iPSC that were previously obtained from patients with familial forms of PD, a severe neurodegenerative disorder [24]. It seems very important to find a possibility for generation of autologous cells for people with chronic diseases, because they are among the first candidates for replacement therapy. Two iPSC lines, iPSC LRRK and iPSC PARK, were obtained from patients with a hereditary form of PD, and one line, iPSC N, was from a healthy donor. We have shown that all the examined lines are able to generate fibroblast-like cells with equal efficiency. It is important to note that generation of derivatives proceeded without using additional mitogens and differentiation factors.

The obtained derivatives der N, der LRRK, and der PARK possessed a fibroblast-like morphology and expressed genes specific for this cell type, including fibronectin, vimentin, type I, III, IV and V collagens (Figure 5). We have established that all three generated lines of derivatives are capable of maintaining the pluripotent state of both autologous and allogeneic iPSC (Figure 9). These data showed that derivatives generated from iPSC of patients with a congenital form of PD, do not differ in their growth and feeder properties, and can be used as a substrate for iPSC growth. The evidence that primary fibroblasts and derivatives can be successfully used for generation of new iPSC lines is already reported in literature [16, 19,20]. In the current study, we were the first to demonstrate that derivatives obtained from cells of a patient with a mutation in the Parkin gene (202-203 AG deletion in the 2nd exon) plus a splicing mutation in the first intron (IVS1+1G/A)) and a patient with mutation in LRRK gene are able to serve as an effective feeder layer for derivation of iPSC. Autologous dermal fibroblasts and classic MEF were used as positive controls. Generation of new iPSC lines was carried out using fibroblasts from a PD patient with a mutation in the GBA gene. Interestingly, after plating transfected fibroblasts

onto prepared feeders, the earliest colonies were observed on autologous dermal fibroblasts followed by allogeneic derivatives, whereas the latest colonies appeared on MEF. At the same time, the effectiveness of the formation of iPSC colonies on MEF was much higher compared to human feeders. Du and colleagues have noted that iPSC colonies cultured on derivatives have a larger diameter than colonies on MEF [19]. In accord with this observation, we noted that iPSC colonies on derivatives were larger than on other substrates. Unfortunately, in the present study we failed to obtain iPSC on autologous feeder - primary fibroblasts. Colonies were formed, but on day 4-5 they started to disintegrate and completely disappeared on day 15. It was probably due to the age of the donor, who was 60 years old. We evaluated proliferative activity of dermal fibroblasts obtained from healthy donors of three age groups (18, 40 and 60 years old). Fibroblasts from young donors and those from middle age donors had a significant advantage in the rate of growth: for these fibroblasts we observed a five-fold increase in the number of cells within 5 days, where as the number of fibroblasts from an elderly donor increased only two-fold (unpublished data). It is also known that production of growth factors, cytokines, chemokines, essential for survival of fibroblasts themselves, as well as for maintenance of iPSC pluripotency, decreases with age. For example, production of collagen in the skin fibroblasts of the elderly (80 years) is reduced by more than 70% compared with young people (18-29 years) [26]. The failure of autologous fibroblasts to provide all the necessary factors to transfected cells for generation of pluripotent colonies can be associated with genetic disorders that led to PD, or individual characteristics of the donor. For better understanding of this process, it might be essential to perform additional studies using multiple lines of primary fibroblasts from patients with genetic disorders, as well as from healthy donors of different age categories. An important feature of derivatives is that, unlike primary fibroblasts, their morphofunctional characteristics are similar to those of fibroblasts derived from newborn foreskin. In this study, we showed that neither the age (over 60 years) of patients, nor the presence of such a severe neurodegenerative disease as PD, is the obstacle for obtaining derivatives from iPSC. All obtained derivatives could serve a feeder layer effectively supporting growth of allogeneic and autologous undifferentiated iPSC. Derivatives generated from iPSC of a patient with autosomal recessive juvenile parkinsonism, that carried compound heterozygous mutations in the Parkin gene (202-203 AG deletion in the 2nd exon) plus a splicing mutation in the first intron (IVS1+1G/A) were able to synthesize all the necessary factors to ensure an effective reprogramming into iPSC of skin fibroblasts from another patient suffering from Parkinson’s disease due to a mutation in the gene encoding the glucocerebrosidase (GBA). Thus, our experiments demonstrated that iPSC from patients with the familial forms of PD could be successfully differentiated into fibroblast-like cells (derivatives). The resulting derivatives can be used not only as an effective feeder layer for propagation of autologous and allogeneic iPSC, but also for generation of new human iPSC lines.

ConclusionThis is the first report, to our knowledge, demonstrating the

effective differentiation of iPSC obtained from patients with




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familial forms of PD (mutations in PARK2 and PARK8 genes) into fibroblast-like cells (derivatives). It was shown that the obtained derivatives can be effectively used as feeder layers not only to maintain the pluripotency of autologous and allogeneic iPSC, but also to obtain new iPSC lines. In future these new patient specific iPSC can be used in cellular transplantation in humans.

AcknowledgementThe authors thank prof. M.A. Lagarkova and prof. S.L. Kiselev

(Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russia) for the providing ES and iPSC cell lines. This study was performed using the equipment of the Center of Cellular and Genomic Technologies of Institute of Molecular Genetics RAS, Moscow, Russia. The study was supported by the grants from the Programs of the Russian Academy of Sciences: “Basic Research for Biomedical Technologies”, “Molecular and Cellular Biology” and the grant from Russian Scientific Fund (N° 14-15-01047).

Conflict of InterestThe authors declare that there are no competing interests

regarding the publication of this paper.

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https://www.ncbi.nlm.nih.gov/pubmed/16904174



































http://journals.plos.org/plosone/article/authors?id=10.1371/journal.pone.0076205




http://www.nature.com/nbt/journal/v22/n1/abs/nbt922.html








































http://stm-journal.ru/en/numbers/2016/4/1293/pdf











fibroblast-like cells derived from ips cells of patients...

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