Differentiation of human fetal mesenchymal stem cells into cells with ...

[Cell Cycle 8:7, 1069-1079; 1 April 2009]; ©2009 Landes Bioscience

Report

IB UT E.

Differentiation of human fetal mesenchymal stem cells into cells with an oligodendrocyte phenotype Nigel L. Kennea,1,2 Simon N. Waddington,6 Jerry Chan,1,3 Keelin O’Donoghue,1,3 Davy Yeung,1,5 Deanna L. Taylor,1,5 Faisal A. Al-Allaf,1,3 Grisha Pirianov,1,3 Michael Themis,6 A. David Edwards,4 Nicholas M. Fisk1,3 and Huseyin Mehmet1,2,* of Reproductive and Developmental Biology; 2Division of Clinical Sciences; 3Division of Surgery, Oncology, Reproduction and Anaesthesia; 4MRC Clinical Sciences Centre; 5Division of Neuroscience and Mental Health; and 6Division of Biomedical Sciences; Faculty of Medicine; Imperial College London; London, UK

ST R

1Institute

DI

Key words: oligodendrocyte, mesenchymal, stem, fetal, differentiation

NO T

This has led to interest into whether exogenous stem cells could be used to repair the injured brain. Stem cells have now been isolated from most adult organs.1 Originally, it was believed that such cells were limited in their differentiation potential to cells of the tissue from which they were derived. This concept has been challenged by research demonstrating cell plasticity in that somatic cells of one tissue type to differentiate into another. For example, NSC can give rise to blood and muscle,2 whilst bone marrow mesenchymal stem cells (MSC) can contribute to non mesoderm-derived tissues including liver, lung, gut and skin.3 Recent studies have indicated that MSC may also have neurogenic potential4-6 and are therefore attractive candidates for neural cell replacement therapy. In studies of cultured human and rodent bone marrow cells treated with retinoids and growth factors, the neuronal (NeuN, βIII tubulin) and astrocyte (Glial Fibrillary Acidic Protein) markers are upregulated7 although no oligodendrocyte specific markers were seen. Other regimens have exposed MSC to reducing agents, β-mercaptoethanol or butylated hydroxyanisole resulting in a rapid adoption of neuronal and astrocytic morphology and phenotype.8 Some investigators have urged caution when interpreting these data, suggesting they may result from vigorous aberrant gene expression rather than ordered gene expression9 or from cell retraction caused by cell stress.10 In vivo work has provided data supporting the neural potential of MSC in both animal models and humans. In murine models of bone marrow transplantation, infused bone marrow cells migrate and integrate into the brain acquiring neuronal markers.11 MSC have been used in the treatment of animal models of various CNS diseases, including Niemann Pick disease (sphingomyelinase deficiency12), multiple sclerosis13 and other inflammatory demyelinating disorders.14 To date, the majority of neural differentiation studies from bone marrow MSC have focused on neurones rather than oligodendrocytes and in the majority of studies, oligodendrocyte markers were not found or examined. Oligodendrocyte replacement would be essential for future cell therapy in many diseases including multiple sclerosis, stroke and perinatal brain injury, in which loss of oligo-

09

Introduction

LA

ND ES

BI

OS CI

EN CE .D O

The potential of mesenchymal stem cells (MSC) to differentiate into neural lineages has raised the possibility of autologous cell transplantation as a therapy for neurodegenerative diseases. We have identified a population of circulating human fetal mesenchymal stem cells (hfMSC) that are highly proliferative and can readily differentiate into mesodermal lineages such as bone, cartilage, fat and muscle. Here, we demonstrate for the first time that primary hfMSC can differentiate into cells with an oligodendrocyte phenotype both in vitro and in vivo. By exposing hfMSC to neuronal conditioned medium or by introducing the pro-oligodendrocyte gene, Olig-2, hfMSC adopted an oligodendrocyte-like morphology, expressed oligodendrocyte markers and appeared to mature appropriately in culture. Importantly we also demonstrate the differentiation of a clonal population of hfMSC into both mesodermal (bone) and ectodermal (oligodendrocyte) lineages. In the developing murine brain transplanted hfMSC integrated into the parenchyma but oligodendrocyte differentiation of these naïve hfMSC was very low. However, the proportion of cells expressing oligodendrocyte markers increased significantly (from 0.2% to 4%) by preexposing the cells to differentiation medium in vitro prior to transplantation. Importantly, the process of in vivo differentiation occurred without cell fusion. These findings suggest that hfMSC may provide a potential source of oligodendrocytes for study and potential therapy.

©

20

The brain has a limited capacity for repair following injury or degeneration and, although endogenous neural stem cells (NSC) proliferate following injury they are inadequate to affect repair. *Correspondence to: Huseyin Mehmet; RY80Y-215; Merck and Co., Inc.; 126 East Lincoln Avenue; P.O. Box 2000; Rahway, New Jersey 07065-0900 USA; Tel.: +1.732.594.2511; Email: [email protected] Submitted: 01/13/09; Accepted: 02/09/09 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/8121

www.landesbioscience.com

Cell Cycle

1069

Oligodendrocyte differentiation of human fetal MSC

©

20

09

LA

ND ES

BI

OS CI

EN CE .D O

NO T

DI

ST R

IB UT E.

dendrocytes or their precursor cells is a major contributor to cerebral damage. In one of the few oligodendrocyte studies, murine bone marrow cells injected in a spinal cord demyelination model appeared to acquire the oligodendrocyte marker, myelin basic protein.15 These data are controversial with other studies demonstrating functional improvement after MSC injection in spinal injury models without neural differentiation.16 In vivo data supporting neural differentiation of bone marrow or purified MSC remains controversial and some results can be explained by cell fusion between transplanted and host cells.17 Others have suggested an alternative explanation for the broader differentiation capacity of bone marrow is the presence of rare populations of more pluripotent cells, and the reports of multipotent adult progenitor cells (MAPC) perhaps adds weight to this argument.18 MAPC can be differentiated to mesodermal and endodermal tissues, and stimulated with growth factors to neurectodermal fates. With neural differentiation protocols, the majority of differentiated MAPC express neuronal neurofilament 200, with only 11% of cells expressing the oligodendrocyte marker myelin basic protein (MBP) on day 10 declining to 2% by day 22.19 Although these oligoFigure 1. Morphological changes in hfMSC treated with oligodendrocyte differentiation medium dendrocyte data are interesting, the low (ODM) and the induction of oligodendrocyte-specific gene transcripts. Following exposure to ODM frequency of differentiation and decline (A) >50% fetal MSC become phase bright and bipolar between 4–7 days, with later maturation in oligodendrocyte number over time into multi-processed cells, resembling mature post-mitotic oligodendrocytes. Nestin, a marker of early would not make them a good model neural progenitors, was upregulated within 2 days of this process, and then declined as demonstrated to study differentiation. Indeed there by immunocytochemistry (B) and RT-PCR (C). Similarly, the neural progenitor markers, Notch-1 and Musashi, were also upregulated 3 days after exposure to ODM and remained throughout differentiation is no readily available source of human (C). RT-PCR analysis demonstrated increased mRNA expression of three transcription factors important oligodendrocyte precursors for study in oligodendrocyte specification Olig1, Olig2 and NKx2.2 (D). A low baseline level of CNPase mRNA or therapy although oligodendrocyte is evident but upregulated with differentiation time. The transcription of the mature oligodendrocyte precursors have recently been isolated protein, MOG, started later on day 9. human fetal brain and adult subcortical white matter and used successfully to remyelinate shiverer mice.20 Results We have identified a population of MSC in the human fetus. Exposure to ODM results in morphological change and acquiThey can be easily isolated from blood, bone marrow and liver. sition of neural progenitor phenotype. Undifferentiated hfMSC These human fetal MSC (hfMSC) are highly proliferative, and we did not express specific early or late markers of oligodendrocyte have previously shown that they readily differentiate into mesoderm21,22 differentiation, but did express low levels of nestin and vimentin. derived tissues such as bone, cartilage, fat and muscle. In Following exposure to ODM there was a dramatic morphological addition, we have found that these cells express pluripotency markers, change within 2–4 days: undifferentiated fibroblast-like cells divide rapidly and have longer telomeres than adult MSC from other 23 acquired a bipolar phenotype with phase-bright nuclei and strongly sources, suggesting that they may display greater plasticity than resembled oligodendrocyte precursor cells (OPC) (Fig. 1A). At 2 adult MSC. This present study describes the acquisition of oligodendays 89 ± 7% of cells strongly expressed nestin with upregulation drocyte phenotype from primary hfMSC both in vitro and in vivo. confirmed by both immunohistochemistry (Fig. 1B) and RT-PCR (Fig. 1C) level. Other markers of early neural development 1070

Cell Cycle

2009; Vol. 8 Issue 7


©

20

09

LA

ND ES

BI

OS CI

EN CE .D O

NO T

DI

ST R

IB UT E.

although CNPase mRNA was expressed in undifferentiated hfMSC, this increased early in the differentiation process (Fig. 1D). MOG expression appeared later at 9 days (Fig. 1D) although myelin proteins could not be detected by immunocytochemical staining. Bipolar cells induced by exposure to ODM expressed the early oligodendrocyte marker A2B5 (Fig. 2A) and NG2 (data not shown), although the proportion of cells varied considerably in different experiments. Increased expression levels of both vimentin, and the oligodendrocyte marker CNPase were detected by western blotting after one week of differentiation (Fig. 2B). Since some studies have suggested that changes in cell morphology during differentiation are due to a stress response,10 cytoskeletal alterations during differentiation of hfMSC into oligodendrocyte-like cells were visualized by F-actin staining with rhodamine phalloidin. F-actin staining suggested that the cytoskeleton did not collapse during differentiation (Fig. 2C, ii) unlike hfMSC treated with 0.125% trypsin EDTA to induce stress (Fig. 2C, iii). The sub-membrane and patchy F-actin staining indicated reorganization of the cytoskeleton rather than retraction of cells due to stress induced by ODM and staining of differentiated cells was similar to F-Actin staining of CG4 OPC cell line (Fig. 2C, iv). Overall, these in vitro data suggested that ODM-exposed hfMSC undergo morphological changes consistent with differentiation along the oligodendrocyte lineage, initially giving rise to cells with phenotypic characteristics of OPC that differentiate into more mature oligodendrocytes with time. Importantly, these data suggest that differentiation may be an ordered process with the acquisition of neural progenitor markers followed by the upregulation of lineage-specific transcription factors (Olig1,2 and Figure 2. Acquisition of oligodendrocyte-specific markers in hfMSC confirmed by NKx2.2). Indeed, by nine days of culture we observed clonal analysis with derivation of neuroectodermal lineage from a single fetal MSC an increase in transcription of mature myelin genes in clone. Following exposure to ODM, the OPC marker A2B5 was upregulated on cultures containing large numbers of multi-processed cells bipolar cells with oligodendrocyte-like morphology (green A). Western blotting resembling mature oligodendrocytes. demonstrated the upregulation of vimentin and CNPase with time of differentiation To further our observations that morphological changes (B). Phalloidin staining of F-actin to show cytoskeletal changes with differentiation are not due to cell stress/retraction. Control hfMSC (C, i), hfMSC exposed to ODM may be due to the plasticity of hfMSC and not to the for 7 days (C, ii), hfMSC exposed to 0.125% Trypsin-EDTA overnight (C, iii), and stress response to the induction factor(s) in the ODM, we the CG4 OPC cell line (C, iv). have also explored the effect of overexpression of Olig2 transcription factor which has been shown previously including Notch 1 and Musashi were also upregulated (Fig. to initiate oligodendrocyte differentiation in NSC.26 We have 1C). Prolonged exposure to ODM resulted in an increase in the constructed a HIV based lentiviral vector for gene transfer and morphological complexity of hfMSC and a subsequent reduction expression of the human Olig2cDNA.27 The production of intact in nestin expression (Fig. 1A and B). virus was confirmed by the presence of the 174 bp amplicon (Fig. Bipolar cells acquired early oligodendrocyte phenotype and 3B upper gel) and exclusion of plasmid DNA contaminants was mature in culture. RT-PCR analysis was carried out on samples confirmed (Fig. 3B lower gel). Western blot analysis confirmed obtained from time-course experiments following exposure to that transduced hfMSC expressed the HIV-Olig2 vector (Fig. ODM for up to 2 weeks. Three transcription factors important 3C). Overexpression of Olig2 gene in hfMSC induced the develin oligodendrocyte specification, Olig1, Olig2 and NKx2.2, were opment of oligodendrocyte-like cells which expressed A2B5 after investigated along with CNPase and the mature oligodendrocyte 48 hours (Fig. 3D, i). Overexpression of Olig2 in human fetal marker myelin oligodendrocyte glycoprotein (MOG). An increase fibroblast cells did not promote oligodendrocyte-like morphology in Olig1, Olig2 and NKx2.2 was observed between 3–6 days and, or A2B5 expression (data not shown). We also explored the role www.landesbioscience.com

Cell Cycle

1071


©

20

09

LA

ND ES

BI

OS CI

EN CE .D O

NO T

DI

ST R

IB UT E.

of the human Nkx2.2 transcription factor by gene transfer. Overexpression of the human Nkx2.2 in hfMSC did not promote oligodendrocyte-like morphology, while co-transduction of Olig2 and Nkx2.2 lead to early cell death (data not shown). Generation of a hfMSC clone. To exclude the possibility of contaminating cells in the hfMSC preparation, we confirmed these findings using a clonal population of cells to demonstrate both mesodermal and neurectodermal differentiation from a single fetal MSC clone. Green fluorescent protein (GFP) expression was used for in vivo cell tracking and we utilized proviral integration site analysis to prove clonality. To express GFP, cells were infected with a HIV-1 based lentiviral vector with a low multiplicity of infection (MOI = 10). Transduction efficiency was 78% with a single infection determined by flow cytometry. For in vivo studies, cultured cells were enriched by fluorescence activated cell sorting to 98% GFP+ with a single sort. We confirmed that GFP+ cells could be expanded in culture without the loss of GFP expression (data not shown). Attempts at culturing single GFP+ cells by limiting dilution, or by micropipette-aided single cell selection did not yield clonal populations when cultured in complete medium or Figure 3. Overexpression of Olig2 transcription factor in human fetal MSC drives oligodendroin conditioned medium from healthy hfMSC cyte differentiation. GFP labelled hfMSC were exposed to ODM for 7 days and stained with cultures. In these experiments single cells were nestin (red; A, i) and the oligodendrocyte marker A2B5 (red; A, ii) Detection of virus producTM visible and survived for several days without tion by RT-PCR (B) Upper gel: RT-PCR reactions were performed using the One-Step RT-PCR kit primed with WPRE primer pair. RT-PCR reaction from negative control (lane 1), HIV-eGFP division. Clones were eventually obtained by vector (lane 2), and HIV-Olig2 vector (lane 3). The 174 bp amplicon generated confirms the + growing single male GFP cells on a feeder layer production of intact viruses. Lower gel: PCR for the corresponding samples to exclude plasmid of senescent female-derived hfMSC grown in DNA contaminants (B). Western blot analysis of transduced hfMSCs with the HIV-Olig2 vector DMEM/10% FBS. Following expansion, GFP+ (lane 2), HIV-eGFP vector (lane 3), non transduced cells (lane 1), and positive control lysate of fluorescence was maintained and cells expressed MO3 cells (lane 4). 50 μg of protein from cell lysates was loaded on the gel and probed with anti-Olig2 (32 kDa) and as a loading control with anti-actin (42 kDa) antibodies (C). 48 hours a male karyotype (XY) with no evidence of after transfection of Olig2, cells were positive for A2B5 (D, i). CG4 cells were used as positive male to female cell fusion. Southern analysis of control (D, ii) untransfected hfMSC were use as negative control (D, iii). digested genomic DNA from a transfected GFP+ clone demonstrated a single integration site confirming clonality. examined immediately following injection and at time points up A clonal population of hfMSC can give rise to oligoden- to 1 month. Injected cells were identified by three methods: GFP drocyte-like cells. To determine whether clonal hfMSC undergo visualization, either directly or using a specific antibody, humanoligodendrocyte and mesodermal differentiation we exposed them specific fluorescence in situ hybridization (FISH), or with the aid both to ODM and differentiation medium for bone. The clonal of human-specific antibodies. For in utero transplantation experipopulation gave rise to cells with oligodendrocyte phenotype with ments, hfMSC were injected into the left frontal horn of the lateral the early expression of nestin (Fig. 3A, i) and A2B5 on bipolar ventricle and cell survival, morphology, migration and phenotype cells (Fig. 3A, ii). The clone could also be differentiated into bone were examined. Immediately following transplantation, naïve confirmed by Von Kossa staining (data not shown). hfMSC were distributed widely throughout the ventricular system. hfMSC in the developing murine brain. To explore oligo- Human cells were easily identified using GFP and FISH (Fig. dendrocyte differentiation of hfMSC in vivo, we employed an 4) and were negative for neuronal and oligodendrocyte markers. embryonic mouse model of brain development. GFP+ hfMSC By 48–96 hours, hfMSC formed aggregates within the cerebral were injected into the cerebral ventricles of embryonic day 16 ventricles, with cells abutting and becoming continuous with the (E16) mouse fetuses in utero. In initial experiments, undifferenti- ventricular wall (Fig. 4B). Cells at this stage expressed vimentin, ated early passage (90% (Fig. 5F) and in no sections did we see colocalization of murine and human signals.


Cell Cycle

1075


OS CI

Methods

©

20

09

LA

ND ES

BI

Isolation and culture of human fetal MSC. Human fetal blood collection was approved by the Research Ethics Committee of Hammersmith and Queen Charlottes and Chelsea Hospital. National (U.K.) guidelines were complied with in the use of fetal tissues for research. hfMSC were isolated as previously described by Campagnoli et al.21 Briefly, nucleated cells were obtained from first trimester fetal blood and resuspended in DMEM supplemented with 10% FBS (Sigma-Aldrich Ltd., Poole, UK) in 6 well plates and cultured at 37°C in 5% CO2. After 72 hours, non-adherent cells were removed and the medium replaced. When 70–80% confluent, cells were trypsinized and subsequently expanded in DMEM with 10% a concentration was used in all subsequent experiments. The oligodendrocyte differentiation medium (ODM) contained a 40% (v/v) mixture of conditioned medium from B104 rat neuroblastoma cells. B104 cells were expanded in DMEM/10% FBS until confluent, then were washed and medium exchanged for serum-free DMEM + N1 supplement (Sigma). After 5 days the medium was removed, centrifuged at 10,000 g for 5 minutes and filtered. This conditioned medium was stored at -20°C until further use. Immunofluorescence staining. HfMSC were plated at 5 x 104/ 2 cm in 8 well chamber slides and exposed to ODM for defined periods of time. Cells were fixed in 4% paraformaldehyde (PFA) 1076

NO T

DI

ST R

IB UT E.

for 10 minutes, washed twice in PBS, and stored at 4°C until use. Fixed cells were permeabilized with 0.2% Triton X-100 (Sigma) for 10 min, and then non-specific protein interactions blocked by 1 hour in 5% normal goat serum/5% bovine serum albumin followed by primary antibody incubation overnight at 4°C. The primary antibodies used were vimentin (Dako Cytomation Ltd., Ely, UK), nestin, NG2, CNPase, βIII tubulin, galactocerebroside (Millipore, Watford, UK), proteolipid protein/DM20, myelin basic protein (Biogenesis, Poole, UK), PDGF receptor α (R&D systems, Abingdon, UK), and hybridoma supernatants O4 and A2B5 (ATCC, Teddington, UK). Negative controls were exposed to the same concentration of isotype control antibody or no primary antibody. Control human brain tissue, or the oligodendrocyte cell line CG4 were used as positive controls. Primary antibodies were used at 1:100–200 dilutions in PBS/0.5% BSA and hybridoma supernatants used diluted 1:10. Following 3 washes, slides were incubated for 1 hour at room temperature with an appropriate biotinylated secondary antibody diluted 1:100 in 1% BSA (Vector, Orton Southgate, UK). Detection of secondary antibodies was with diluted (1:100) streptavidin-fluoresceine or streptavidin-TexasRed (Vector) or with ABC followed by 3,3' diaminobenzidine (DAB, Vector). Immunostained cells were then washed three times in PBS and, in fluorescence experiments, the nuclei were counterstained with DAPI. F-actin cytoskeleton visualization. To determine whether the neuronal morphology observed after ODM treatment was due to collapse of the F-actin or reorganization of the cytoskeleton, cells were labelled with rhodamine phalloidin. Cells were fixed in 4% PFA for 10 minutes, washed twice in PBS, and then incubated with the rhodamine phalloidin (1:300; Sigma) for 20 minutes at room temperature. Cells were counter stained with DAPI and mounted. As a positive cell stress control, hfMSC were treated with 0.125% Trypsin EDTA (Sigma) overnight at 37°C. Images were analyzed using a Nikon, Eclipse E600 fluorescence microscope. Reverse transcriptase polymerase chain reaction. Total RNA was isolated with TRIzol Reagent (Invitrogen Ltd., Paisley, UK) using manufacturer’s instructions, and quantified spectrophotometrically. 20 μg samples were reverse transcribed using oligo-(dT)12-18 primer and the SuperScript II Kit (Invitrogen Ltd.,). Parallel samples were generated with omission of reverse transcriptase in the reaction mix to rule out genomic DNA contamination. Amplification of the control RNA without reverse transcription did not generate any products in PCR reactions (data not shown). Primers were designed from published cDNA sequences and custom made (Invitrogen Ltd.,), conditions were optimized for each primer pair (Table 1). Following amplification (25–35 cycles of 1 minute at 94°C, 1 minute at 58°C and 1 minute at 72°C) in a Techne Genius thermal cycler (Techne, Duxford, UK), the PCR products were resolved on 1–1.5% agarose gels and products photographed under UV light. Product size was defined using Bioline Hyperladder IV (Bioline, London, UK). GAPDH was a loading control, and embryonic human fetal brain cDNA was used as a positive control. Western blotting. At defined time points, cells were washed with cold HBSS and collected (by scraping) and centrifuged.

EN CE .D O

the ventricles, hfMSC migrated into the parenchyma. In contrast to the experiments using undifferentiated cells, we found clear acquisition of NG2 within 48 hours and then cells positive for A2B5, CNPase and MBP by 7 days. Over the last few years, transdifferentiation of MSC cells into neural cell types has disputed. Although, most of the debated were generated from studies of bone marrow-derived MSC. Recent evidence supports our finding that the fetal blood-derived MSC possess the potential to differentiate into neural lineage.40 Moreover, recent studies have demonstrated that bone marrow MSC can differentiate into functioning Schwann cells41,42 which further indicate the potential of hfMSC becoming myelinating cells. Further work is underway to investigate if the hfMSC derived oligodendrocyte-like cells are functional and whether the cell environment particularly following injury could further enhance oligodendrocyte differentiation. The mechanisms underlying the in vivo morphological and phenotypic changes of hfMSC remain unknown. We have shown with clonal experiments that this differentiation is not due to rare contaminating more pluripotent cells, and our in vivo work suggests it does not appear to be cell fusion. Whether de-differentiation, or transdifferentiation of hfMSC to a more pluripotent cell, or whether these cells in the fetal circulation are more pluripotent per se warrants further investigation. hfMSC are clearly a promising source of pluripotent cell for study and potential therapy. In addition to the neural potential of the hfMSC cells, transplantation of these cells may have neuroprotective effects and/or immune modulation properties as observed in other MSC.43 We believe that hfMSC will play an important role in various white matter injury in the coming future.

Cell Cycle

2009; Vol. 8 Issue 7


Table 1 PCR primers Antisense primer

Product (kb)

TCC AGG AAC GGA AAA TCA AG

TAGAGA CCT CCG TCG CTG TT

388

Notch1

TCA ATG AGT TCC AGT GCG AG

AGG TGT AAG TGT TGG GTC CG

137

Musashi

GGT TTC CAA GCC ACA ACC TA

GAG GAA TGG CTG TAA GCT CG

145

Olig2

TAA AAG GCA GTT GCT GTG GA

GAC GCT ACA AAG CCC AGT TT

286

Olig1

GAG GTC ATC CTG CCC TAC TC

CTG CCC AGC AGT AGG ATG TA

113

NKx2.2

TTA CAG AAT GTT TGC GCA GC

AAC CCA AAC AAG CCA CAA AG

416

AAC TGA CCC TCC CTT CCT GT

GGT AAA TAG CCC CAG CCT TC

523

TTG GTG AGG GAA AGG TGA CT

TCA AAA GTC CGG TGG AGA TT

280

hosphatase (Promega) treated pRRL-SIN-cPPT-PGK-eGFP. p WPRE viral plasmid. DNA ligations were performed using T4 ligase (Invitrogen). Vesicular stomatitis virus G (VSV-G) pseudotyped recombinant lentiviral vectors were generated by standard three plasmid co-transfection of human embryonic kidney (HEK) derived 293T cells. The self-inactivating transfer vector plasmid encoding the Olig2 (pRRL-SIN-cPPT-PGK-Olig2-IRES-dsRed2-WPRE) or the Nkx2.2 (pRRL-SIN-cPPT-PGK-Nkx2.2-IRES-eGFP-WPRE), or the control vector encoding eGFP (pRRL-SIN-cPPT-PGK-eGFPWPRE), the packaging plasmid pCMVR8.91, and the envelope plasmid (pMDG) were used to generate vector particles. Four million 293T cells were seeded in one 10 cm dish overnight prior to transfection. Cells were cultured in Dulbecco modified Eagle medium (DMEM) with 10% fetal calf serum (FBS) in a 5% CO2 incubator at 37°C. A total of 10 μg plasmid DNA was used for the transfection of a single dish: 2 μg of the envelope plasmid, 4 μg of packaging plasmid, and 4 μg of transfer vector plasmid. This plasmid mixture was complexed with 3 μl FuGene6 (Roche) per 1 μg DNA in 1 mL Optimem (Invitrogen) at room temperature for 45 minutes. The DNA/FuGene6 complexes were then added to the cells. After overnight incubation at 37°C in a 5% CO2 incubator, the medium was replaced by fresh DMEM supplemented with 10% FBS or with N1 supplement and 40% B104 condition medium. At 60 hours and 72 hours after transfection the medium was harvested, cleared by low-speed centrifugation (1,200 rpm, 5 minutes), and filtered through a 0.22 μm filter. Vector particles carrying the eGFP gene used freshly without further concentration. The hfMSC were transduced with a multiplicity of infection (MOI) of 10, and after 12 hours the medium exchanged for fresh growth medium. Vector particles carrying Olig2 or Nkx2.2 gene were concentrated 500 to 1,000-fold by ultracentrifugation at 50,000 g for 2 hours at 4°C. The pellet was resuspended in serumfree medium and used freshly to transduced the cells. Transfected hfMSC were allowed to recover in DMEM supplemented with 10% FBS for 24 hours and then the medium was replace with ODM supplemented with 5 ng/ml FGF and 1 ng/ ml PDGF. Viral RNA precipitations, extraction and DNAse I treatment. 1.2 ml of 0.22 mm filtered unconcentrated viral stock from producer

©

20

09

LA

ND ES

BI

OS CI

EN CE .D O

Cell pellets were resuspended in 1% SDS (90°C) and heated for a further 5 minutes. Resulting lysates were centrifuged at 10,000 g at 4°C for 5 minutes and the supernatant collected for western analysis. Protein concentrations were calculated by the BCA method (Pierce Chemical Co., Cramlington, UK). Equal amounts (50 μg) of protein were loaded onto 10% acrylamide gels and resolved proteins were transferred onto polyvinylidine difluoride (PVDF) membranes (Millipore). Non-specific binding on PVDF membranes was blocked by incubation at room temperature for 1 hour in 5% non-fat dry milk powder in PBS containing 0.1% Tween 20 (PBST). Membranes were then incubated overnight at 4°C with primary antibodies diluted 1:1,000 in PBST containing 5% non-fat dry milk, washed with PBST, and incubated for 1 hour at room temperature with a 1:1,000 dilution of horseradish peroxidase-conjugated secondary antibody (Amersham, Buckinghamshire, UK) in PBST containing 5% non-fat dry milk. After washing with PBST, detection was with enhanced chemiluminescence (ECL kit, Amersham). The antibodies used were to vimentin (Dako) and CNPase (Millipore) with α-tubulin as a loading control. Preparation of Olig2/Nkx2.2 viral vectors. Two HIV based vectors were constructed. The pRRL-SIN-cPPT-PGK-Olig2IRES-dsRed2-WPRE vector expressing the Olig2 gene and the pRRL-SIN-cPPT-PGK-Nkx2.2-IRES-eGFP-WPRE vector expressing the Nkx2.2 gene. Transgene expression in both vectors is driven by the phosphoglycerate kinase (PGK) promoter. For construction of the pRRL-SIN-cPPT-PGK-Olig2-IRESdsRed2-WPRE vector, the 5'-Olig2-IRES-DsRed-3' cassette was excised from the pOlig2cDNA-IRES2-DsRed2 expression plasmid using the NheI and the MfeI restriction enzymes (Invitrogen), blunt ended with klenow large fragment polymerase I (Roche, Basel, Switzerland) and then ligated to BamHI/Sal I digested, blunt ended and alkaline phosphatase (Promega, Southampton, UK) treated pRRL-SIN-cPPT-PGK-eGFP.WPRE viral plasmid. The ligation was performed using T4 ligase (Invitrogen). For construction of the pRRL-SIN-cPPT-PGK-Nkx2.2-IRES-eGFPWPRE vector, the 5'-Nkx2.2-IRES-eGFP-3' cassette was excised from the pNkx2.2cDNA-IRES2-eGFP expression plasmid using the NotI and the XhoI (Invitrogen) restriction enzymes, blunt ended with klenow large fragment polymerase I (Roche) and then ligated to BamHI/Sal I digested, blunt ended, and alkaline

ST R

MOG

DI

CNPase

IB UT E.

Sense primer

Nestin

NO T

PCR primer


Cell Cycle

1077


NO T

DI

ST R

IB UT E.

very high GFP expression were gated out so as not to enrich for cells with many viral integration sites. Generation of single cell clones. The culture of single GFP+ cells in 96 well plates, or in NunclonTM MicrowellTM Plates (volume of 5–10 μl), did not yield clonal populations when cultured in complete medium or conditioned medium from healthy hfMSC cultures. In these experiments single cells were visible and survived for several days without division. Clones were obtained by growing single male GFP+ cells on a feeder layer of senescent female hfSC in DMEM/10% FBS. Male GFP+ colonies were cultured in 96 well plates and then expanded through 24, 12 and then 6 well plates. Southern blot analysis was used to determine whether the transduced hfMSC clone contained single or multiple proviral insertions and to gain an indication of clonality. The method used was that previously described.12 Briefly, 10 μg of genomic DNA was digested with the PstI restriction enzyme (present as a unique site in the vector backbone) and fragments were separated on agarose gels before transfer to Nylon membranes (Hybond-NTM). A 600 bp probe was generated by Not I digestion of the vector representing the woodchuck post-transcriptional regulatory element (WPRE, also present on the vector) which was gel purified and labelled using a random primer labelling kit (Mega-primeTM system Amersham) according to the manufactures instructions with 50 mCi of α-32P-CTP (3,000 Ci/mM) to generate a probe with specific activity of 109 cpm/mg. A Bio-SpinTM 6 column (Bio-Rad) was used to remove unincorporated nucleotides. Following hybridization overnight the membrane was washed as required and the membrane exposed to Kodak XAR-2 X-ray film. Differentiation of clonal hfMSC. Oligodendrocyte differentiation was performed clonal GFP+ cells. In addition, to determine whether cells had retained mesodermal differentiation potential, osteogenic differentiation was assessed by incubating the cells with DMEM with 10% FBS supplemented with 10-8 M dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate for 2 weeks as previously described.25 Bone mineralization was confirmed by Von Kossa staining. Intracerebral injections of hfMSC. Pregnant female MF1 mice (Harlan UK, Bicester, UK) at 15–16 days gestation were used in this study. All procedures were carried out in accordance with the Animal Scientific Procedures Act (1996) and under Home Office approval. Under isofluorane anaesthesia, the uterus was exposed through a full-depth midline laparotomy. Cells 5 x 104 (in 5 μl DMEM) were injected into the anterior horn of the lateral ventricle on the left side via trans-uterine injection using a 33-gauge needle. Up to six fetuses were injected per dam. The laparotomy was closed and the mouse permitted to recover in a warm cage. For endpoint analysis, mice were anaesthetized with isofluorane and perfusion-fixed with 4% PFA. Brains were removed and prepared for frozen sectioning, or fixed overnight in PFA at 4°C prior to paraffin embedding. 5 μm sections were cut for analysis. The fate of transplanted cells was examined immediately following injection and at time points up to 4 weeks. Fluorescence in-situ hybridization (FISH). Paraffin or frozen sections were fixed with either 100 μl of 3:1 v/v methanol:

©

20

09

LA

ND ES

BI

OS CI

EN CE .D O

cell supernatants were added into a pre-cooled 1.5 ml Eppendorf tube and topped up with pre-cooled 0.3 ml of 20% polyethylene glycol (PEG 8,000) in 2.5 M NaCl. The tube was inverted twice to mix the solutions and incubated for 1 hr at 4°C to precipitate the viral RNA. The mixture was then centrifuged at 14,000 rpm for 30 min at 4°C. The viral RNA pellet was then resuspended in 140 μl of DEPC-treated H2O and used for RNA extraction. Viral RNA was isolated from viral particles using a QIAampTM Viral RNA mini kit (QIAGEN, Crawley, UK) following the manufacturer’s instructions. RNA was isolated from 140 μl of precipitated viruses or from 10 μl of concentrated viruses. In brief, the sample is first lysed under highly denaturing conditions to inactivate RNases and to ensure isolation of intact viral RNA. The buffering condition was then adjusted to provide optimum binding of the RNA to the QIAampTM membrane. The RNA binds to the membrane and contaminants are washed away. Pure RNA was then eluted with 60 μl special RNase-free buffer. The Ambion DNA-freeTM kit (Ambion, Warrington, UK) was used to remove DNA contamination with isolated viral RNA, according to the manufacturer’s instructions. Approximately 20 μl of isolated viral RNA was added into an Eppendorf tube and incubated for 30 min at 37°C with 5 μl of 10x DNAse I reaction buffer, 22 μl of nuclease-free water and 3 μl of (2 U/μl) DNAse I. Inactivation of the enzyme and removal of divalent cations was achieved by adding 10 μl of the DNAse I inactivation reagent. The tube was incubated for 2 min at room temperature and pulse centrifuged for 30 sec to pellet the DNAse inactivation reagent. Supernatant containing 50–55 μl DNA-free viral RNA was transferred into a new Eppendorf tube and stored at -70°C for RT-PCR analysis. Detection of virus production by RT-PCR. The One-Step RT-PCRTM kit (QIAGEN) was used, following the manufacturer’s instructions, for qualitative determination of virus production. For detection of virus production,