Original Article

Generation of Human b-Thalassemia Induced PluripotentCell Lines by Reprogramming of Bone Marrow–Derived

Mesenchymal Stromal Cells Using Modified mRNA

Ioanna Varela,1,5 Angeliki Karagiannidou,1 Vasilis Oikonomakis,2 Maria Tzetis,2 Marianna Tzanoudaki,3

Elena-Konstantina Siapati,4 George Vassilopoulos,4 Stelios Graphakos,1

Emmanuel Kanavakis,2,5 and Evgenios Goussetis1

Abstract

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfullyin reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method onbone marrow–derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with b-thalassemia (b-thal) with the aim to generate trangene-free b-thal-iPSCs. Transfection of 104 BM-MSCs by lipofection withmRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of fiveiPSC colonies, from which three were picked up and expanded in b-thal-iPSC lines. After 10 serial passagesin vitro, b-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH)and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profilecompared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas itdiffers from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineageshowed the generation of CD34 + progenitors up to 10%, but with a decreased hematopoietic colony-formingcapability. In conclusion, we report herein the generation of transgene-free b-thal-iPSCs that could be widelyused for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-basedreprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

Introduction

The in vitro reprogramming of somatic cells to plur-ipotency has allowed the generation of disease- and

patient-specific pluripotent stem cell lines that are a valuablemeans for studying disease pathophysiology and for devel-oping effective target therapies (Park et al., 2008; Takahashiet al., 2007). Several induced pluripotent stem cell (iPSC)lines have been generated from patients with genetic hema-tologic diseases, including Fanconi anemia (Muller et al.,2012), sickle cell anemia (Zou et al., 2011), Diamond–Blackfan anemia (Garcon et al., 2013), b-thalassemia (b-thal)(Fan et al., 2012; Wang et al., 2012; Ye et al., 2009a), andothers. Regarding b-thal, one of the most common hereditary

anemias, disease-specific iPSC lines have been used for thegeneration of gene-corrected cells with potential applicationsin clinical cell therapy protocols (Papapetrou et al., 2011;Wang et al., 2012). Such protocols would include the fol-lowing steps: (1) Generation of patient-specific iPSCs; (2)gene correction of the disease-causing mutations and gener-ation of gene-corrected iPSC lines; and (3) differentiation ofthe corrected iPSCs into hematopoietic stem cells. Most of b-thal-iPSC lines were generated from reprogramming of hu-man fibroblasts by forced ectopic expression of definedtranscription factors, such as Oct4, Sox2, Klf4, and c-Myc,using viral or other DNA-based technologies.

However, all methods that use DNA constructs carry therisks associated with the integration of exogenous genetic

1Stem Cell Transplant Unit, Aghia Sophia Children’s Hospital, Athens, 11527, Greece.2Department of Medical Genetics, Medical School, University of Athens, Athens, 11527, Greece.3Department of Immunology - Histocompatibility, Aghia Sophia Children’s Hospital, Athens, 11527, Greece.4Division of Genetics and Gene Therapy, Center for Basic Research II, Biomedical Research Foundation of the Academy of Athens,

Athens, 11527, Greece.5Research Institute for the Study of Genetic and Malignant Disorders in Childhood, Aghia Sophia Children’s Hospital, Athens, 11527, Greece.

CELLULAR REPROGRAMMINGVolume 16, Number 6, 2014ª Mary Ann Liebert, Inc.DOI: 10.1089/cell.2014.0050

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material into the genome of the target cells. Thus, the mostpromising reprogramming methods for generating iPSCssuitable for clinical application are those that use eitherprotein or mRNA or micro RNA (miRNA) molecules. Al-though induction of pluripotency in human cells by directdelivery of four reprogramming proteins (Oct4, Sox2, Klf4,and c-Myc) or by transfection of mature miRNAs has beenreported, the generation of iPSCs by both strategies was veryslow and inefficient (Kim et al., 2009; Miyoshi et al., 2011). Incontrast, the delivery by lipofection of synthetic mRNAmolecules encoding reprogramming transcription factorshas been shown to be highly effective in reprogramming ofhuman fibroblasts in terms of high reprogramming efficiencyand rapid kinetics with which iPSCs were generated. Morespecifically, the mRNA-based reprogramming protocol de-veloped by Warren et al. (2010) is two times faster and 35-fold more efficient in reprogramming human fibroblasts thanthe viral one. Nevertheless, how efficient this method is onother cell types such as bone marrow–derived mesenchymalstromal cells (BM-MSCs) remains to be shown. In the presentstudy, we aimed at generating transgene-free b-thal-iPSClines through reprogramming of BM-MSCs by syntheticmodified mRNA molecules.

Materials and Methods

Cell culture

BM was harvested from the posterior iliac crest foran autologous backup BM graft of a patient with b-thalmajor (IVSI-n6T/C/ - 87C/T, HBB:c.[92 + 6T/C] +[-137C/T]), who would undergo hematopoietic stem celltransplantation from her human leukocyte antigen (HLA)-matched sibling. A sample of 15 mL was used after writteninformed consent from the parents. The research protocolwas approved from the Ethical Committee of the AghiaSophia Children’s Hospital (Athens, Greece). Mononuclearcells were isolated from BM by density gradient centrifu-gation on Ficoll and suspended in Dulbecco’s ModifiedEagle Medium (DMEM; GlutaMAX, Invitrogen, Grand Is-land, NY, USA), supplemented with 10% fetal bovine serum(FBS; Stemcell Technologies, Vancouver, BC, Canada) and20 ng/mL basic fibroblast growth factor (bFGF; R&D Sys-tems, Minneapolis, MN, USA), and placed into a 75-cm2

flask. The adherent cells were proliferated and passaged twotimes to obtain a population enriched in MSCs. Culturedcells were analyzed by flow cytometry (Epics XL-MCL,Beckman Coulter Inc.) and were stained with the followinghuman specific monoclonal antibodies: CD29-FITC, CD90-PE, CD44-FITC, CD105-FITC, CD45-PC5, and CD31-PE.

BJ human foreskin fibroblasts were purchased from Stem-gent (Cambridge, MA, USA) and human neonatal foreskinfibroblasts (NuFF) were obtained from GlobalStem (Gai-thersburg, MD, USA). Skin fibroblasts were maintained inDMEM GlutaMAX containing 10% FBS.

Generation of iPSCs with synthetic mRNA

Synthetic mRNAs (Stemgent, Cambridge, MA, USA) offive transcription factors, Oct4, Klf4, Sox2, c-Myc, and Lin28(in a stoichiometry of 3:1:1:1:1, respectively), were used forthe reprogramming of BM-MSCs to pluripotency. ThesemRNAs incorporate modifications designed to overcome

innate antiviral responses. The mRNA reprogramming wasperformed according to the manufacturers’ recommenda-tions. Briefly, NuFF cells were irradiated (40 Gy) and see-ded at 2.5 · 104 /cm2. The following day, BM-MSCs wereplated to a density of 2500/cm2 in duplicate cultures. After 2days, the culture medium was replaced with Pluriton me-dium (Stemgent) that was supplemented with 200 ng/mLB18R interferon inhibitor (eBioscience, San Diego, CA,USA), and mRNA transfections were initiated with RNAi-MAX Transfection Reagent (Invitrogen) cationic lipid de-livery vehicles and repeated every 24 h for 18 days. Pluritonmedium was replaced daily, 4 h after transfection. The dailyRNA dose applied was 1200 ng per well (six-well plateformat). mRNA encoding nucleus-localizing green fluores-cent protein (nGFP) was also included in the mRNA cock-tail as a marker for monitoring transfection efficiency.Moreover, a GFP mRNA dose of 500 ng was delivered to awell of a 12-well plate using RNAiMAX Trasnfection Re-agent to test the transfection efficiency for BM-MSCs. Forthe positive control, we performed a reprogramming ex-periment with BJ human fibroblasts at the same time.

Culture and expansion of iPSCs

Each iPSC colony was picked manually and transferredto an individual well of a 12-well plate precoated withMatrigel (BD Biosciences, Franklin Lakes, NJ, USA) inmTeSR medium (Stemcell Technologies). For the first fewpassages, the cells were passaged mechanically and furtherexpanded by enzymatic passaging with dispase (StemcellTechnologies).

Human ESC line

The HUES-9 cell line was used in this work and served asa positive control in comparative molecular and culturestudies assessing pluripotency and differentiation potentialof iPSCs, respectively. HUES-9 cells were grown on platesprecoated with Matrigel in mTeSR medium.

Flow cytometry analysis for expressionof pluripotency markers

Oct3/4 and stage-specific embryonic antigen-4 (SSEA-4)primary antibodies (Stemcell Technologies) were used tocharacterize iPSCs by flow cytometry. Staining with uncon-jugated mouse isotypic controls was performed in parallel.The secondary antibody used was fluorescein isothiocyan-tate (FITC)-conjugated goat anti-mouse immunoglobulin M(IgM). Briefly, for surface SSEA-4 staining, cell were firstincubated in a final 1:10 dilution of primary antibody for30 min, subsequently washed and blocked with 10% FBS/phosphate-buffered saline (PBS), and, finally, incubatedwith secondary antibody for 30 min. For intracellular Oct3/4staining, IntraPrep Permeabilization Reagents (BeckmanCoulter Inc., Nyon, Switzerland) were additionally used.Namely, iPSCs were first fixed in paraformaldehyde solutionfor 15 min at room temperature. After washing with 10%FCS/PBS, cells were initially permeabilized in saponin so-lution and labeled with the primary antibody for Oct3/4, at afinal dilution of 1:10. After incubation for 30 min, cells werewashed in 10% FBS/PBS and incubated with saponin solu-tion and a final dilution of 1:10 of the secondary antibody.

2 VARELA ET AL.

Samples were suspended in PBS and analyzed by flow cy-tometry (FC500, Beckman Coulter Inc.).

RT-PCR analysis

Total mRNA was extracted from iPSC pellets using anRNeasy Plus Mini-kit (Qiagen GmbH, Hilden, Germany) andreverse transcribed into cDNA using a SuperScript ReverseTranscriptase Kit (Invitrogen). The resulting cDNA was thenused as a template for detecting pluripotency marker geneexpression of OCT3/4, REX-1, SOX2, and NANOG. Resultswere compared to expression levels of housekeeping geneGAPDH (glyceraldehyde 3-phosphate dehydrogenase).

DNA array comparative genomic hybridization analysis

Total genomic DNA was extracted with Qiagen BloodMini-kit. 800ng of the extracted gDNA was amplified andlabeled using the SureTag DNA Labeling Kit, Two Colors(Agilent Technologies, Santa Clara, CA, USA). The labeledDNA was hybridized to Agilent SurePrint G3 Human4x180k CGH + SNP microarrays. The 4x180K CGH + SNPplatform is composed of 110,712 [comparative genomichybridization (CGH)] + 59,647 [single-nucleotide polymor-phism (SNP)] 60-mer oligonucleotide probes with 25.3-kboverall median probe spacing (5-kb in ISCA regions). AgilentFeature Extraction Image Analysis Software (v. 10.7.3) wasused to extract data from raw microarray image files. Datavisualization and analysis was performed using Agilent CytoGenomics (v. 2.7) software. For the location of genes[annotated against National Center for Biotechnology In-formation (NCBI) build 37, hg19] in the deleted/duplicatedgenomic segments the University of California Santa Cruz(UCSC; http://genome.ucsc.edu/) and the Database of Geno-mic Variants (http://projects.tcag.ca/variation/) were used.

Gene expression analysis

Total RNA was extracted with a Qiagen RNeasy MiniKit. A 25-ng amount of total RNA was amplified and la-beled using the Agilent Low Input Quick Amp Labeling Kit,One-Color. The labeled RNA was hybridized to AgilentSurePrint G3 Human Gene Expression 8x60K Microarrays.The specific platform is composed of 50,599 60-mer oli-gonucleotide probes. Agilent Feature Extraction ImageAnalysis Software (v. 10.7.3) was used to extract data fromraw microarray image files. Data visualization and analysiswas performed using GeneSpring GX (v. 11.0) software.

In vitro differentiation

For embryoid body (EB) formation, iPSCs were treated with1 mg/mL dispase and transferred to six-well low-attachmentplates in differentiation medium composed of knockoutDMEM supplemented with 20% knockout serum replacement,1 · nonessential amino acids, 1 mM l-glutamine, and 0.1 mMb-mercaptoethanol. The medium was changed every other day.After 7 days, the formation of EBs was examined.

In vivo teratoma formation

For the teratoma formation assay, iPSCs from one T25flask were harvested by dispase treatment (1 mg/mL) andresuspended in DMEM/F12 and Matrigel at a ratio 2:1. The

cell mixtures were subcutaneously injected into 6-week-oldsevere combined immunodeficient (SCID) mice. After 7–11weeks, the resultant tumors were dissected, fixed in 4%paraformaldehyde, embedded in paraffin, and sectioned.After staining with Hematoxylin & Eosin, the sections wereexamined for the presence of tissues derived from the threegerm layers.

Hematopoietic differentiation

The hematopoietic differentiation of BM-MSCs wasinvestigated in duplicate adherent cell cultures using therecombinant protein-based, animal product-free mediumSTEMdiff APEL (Stemcell Technologies). Briefly, 3 daysafter passaging iPSCs (day 0), mTeSR medium was replacedwith STEMdiff APEL medium, supplemented with 30 ng/mLvascular endothelial growth factor (VEGF), 30 ng/mL bonemorphogenetic protein-4 (BMP-4), 40 ng/mL stem cell factor(SCF), and 50 ng/mL Activin A (all from R&D Systems). Onday 4, this medium was removed and replaced with STEM-diff APEL medium supplemented with 300 ng/mL SCF,300 ng/mL Flt-3 ligand, 10 ng/mL interleukin-3 (IL-3), 10 ng/mL IL-6, 50 ng/mL granulocyte colony-stimulating factor(G-CSF), and 25 ng/mL BMP-4 (all from R&D Systems).The medium was changed every 3 days. On day 13, cellswere treated with TrypLE Select (Invitrogen) and analyzedfor expression of hematopoietic markers CD34 and CD45 byflow cytometry. Single cells were also cytospun on slides andstained with May Gruenwald–Giemsa. For the colony form-ing unit (CFU) assay, 1–2 · 105 cells were plated in duplicate35-mm tissue culture dishes containing 1 mL of MethoCultH4230 (Stemcell Technologies) supplemented with 100 ng/mL SCF, 5 U/mL erythropoietin (EPO), 50 ng/mL IL-3,50 ng/mL G-CSF, 50 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems) and cultured for13 more days.

Results

Generation of b-thal-iPSCs from BM-MSCs

We first established a culture of BM-MSCs obtained fromthe thalassemic patient as described in Materials and Meth-ods. After two passages in vitro, the cells displayed a fibro-blast-like morphology. Flow cytometric analysis of culturedcells demonstrated expression of cell-surface markers typicalof cultured MSCs, such as CD44, CD105, CD29, and CD90,and did not express the markers CD45 and CD31 specific forhematopoietic and endothelial cells, respectively.

To reprogram these cells, MSC cultures containing 104

cells were transfected daily with a five factor–modifiedmRNA cocktail for 18 days. Loosely packed colonies beganto emerge as early as day 11 of transfection, whereas typicaliPSC colonies could be seen at day 15. These coloniesreached the optimum size to be picked up around days 18–21. An outline of the reprogramming approach is shown inFigure 1A. To test the transfection efficiency of mRNAs forBM-MSCs, we analyzed the GFP expression by flow cy-tometry. Twenty-four hours posttransfection, we detected85% GFP-positive cells (Fig. 1B). To compare the efficiencyof iPSC derivation from BM-MSCs directly, we performed anexperiment in which the reprogramming efficiency of thismethod was also defined in BJ fibroblasts. The average

GENERATION OF b-THAL-iPSCs USING mRNAs 3

number of PSC-like colonies developed from two wells wasfive for BM-MSCs and about 150 for BJ fibroblasts, corre-sponding to a reprogramming efficiency of 0.05% and 3.7%,respectively. These results demonstrate a 74-fold lower iPSCconversion efficiency in BM-MSCs than in BJ fibroblasts.After 2 days posttransfection, when the BM-MSC–derivediPSC colonies had a good-looking PSC-like morphology andwere sufficiently expanded in size, we manually picked threecolonies and replated each colony to an individual well pre-coated with Matrigel in mTeSR medium for maintenance in afeeder-free, xeno-free system. All three clones were expandedin iPSC lines and retained their undifferentiated state formore than 3 months in culture.

Phenotypic and molecular characterizationof b-thal-iPSCs

A number of assays have been applied to detect plurip-otent markers of the b-thal-iPSC lines that have been gen-erated from BM-MSCs. All analyses were performed onsamples obtained from passage (P) 10. Flow cytometryanalysis revealed that b-thal-iPSC lines express high levelsof the intracellular antigen Oct3/4 and the surface antigenSSEA-4 (Fig. 2A). RT-PCR results showed that b-thal-iPSCs express the pluripotency markers OCT3/4, REX-1,SOX2, and NANOG at levels equivalent to HUES-9 ESCs,whereas the original BM-MSCs had only low expression of

OCT3/4 and NANOG (Fig. 2B). To confirm that the gen-erated iPSCs were derived from the patient’s cells, weconfirmed the b-thal genotype for both BM-MSCs and de-rived iPSCs. When global gene expression was comparedusing Agilent microarrays analyzing the totality of knownhuman transcripts, iPSC clones showed similar pattern toHUES-9 cells but not to parental BM-MSCs. Specifically,pluripotency-associated transcripts were clearly upregulatedin the iPSCs compared to the parental BM-MSCs to levelscomparable to hESCs (Fig. 2C).

To ensure that the reprogramming process did not resultin any genetic alteration in the cells, we analyzed samplesbefore the first passage of iPSCs with DNA array CGH(aCGH). We performed the same analysis at P10, to deter-mine whether culture adaptation caused chromosomal ab-errations in cells. Molecular karyotypes of iPSC lines werenormal, and no genetic alteration was revealed in compari-son to the original BM-MSCs (Fig. 3A).

In vitro differentiation and in vivo teratoma formation

Pluripotency was further confirmed both in vitro andin vivo by the formation of EBs and development of tera-tomas in SCID mice, respectively. After 7 days of culture inlow-attachment plates, iPSCs formed EBs, indicating suc-cessful differentiation into the three germ layers in vitro(Fig. 3B). When b-thal-iPSCs were injected subcutaneously

FIG. 1. Transfection of human thalassemia BM-MSCs with modified mRNA. (A) Schematic representation of the re-programming approach using modified mRNAs showing morphological changes observed during the experiment. Detectionof nGFP-expressing cells by immunofluorescence 16-h posttransfection on day 1. An area of morphological changecharacteristic for the mesenchymal-to-epithelial transition observed on day 11 is marked with a dashed circle. Magnifi-cation,10 · . (B) Percentage and median fluorescence intensity (MFI) of GFP expression in BJ fibroblasts and BM-MSCs24 h after transfection with mRNA encoding GFP (a). Flow cytometry histograms show GFP expression in BJ (b) and BM-MSCs (c).

4 VARELA ET AL.

into immunodeficient mice, we observed teratoma formationafter 7–11 weeks. Histological examination revealed that theteratomas contained tissues derived from all three germlayers, including intestinal epithelium (endoderm), cartilage(mesoderm), and neuroepithelium (ectoderm) (Fig. 3C).

In vitro hematopoietic differentiation

To investigate the ability of the b-thal-iPSCs to differ-entiate into hematopoietic progenitor cells, we used afeeder-free protocol based on the animal product–free me-dium STEMdiff APEL supplemented with growth factors,as detailed in Materials and Methods, in adherent cell cul-ture, on a Matrigel-coated surface. For the experiments,iPSCs were used at P10. By day 8, we observed the for-mation of areas consisting of single small round cells onadherent cells (Fig. 4A). On day 13, cells were harvestedand analyzed by flow cytometry. CD34 expression was de-tected up to 10% of the total cells, of which the majorityrevealed a dim CD45 co-expression, whereas only a minorcell population showed CD45 bright expression (Fig. 4B).Their hematopoietic potential was further examined by

methylcellulose cloning assays. We observed a reducedclonogenic potential with the development of three to fivegranulocyte-monocyte colonies (GM-CFU)/105 cells, whereaserythroid colonies could not be detected. In contrast, using thesame culture protocol, HUES-9 cells differentiated efficientlyinto CD34+CD45+ hematopoietic progenitors displayingnormal colony-forming activity for both erythroid and granu-lomonocytic lineages.

Discussion

With the aim of generating b-thal-iPSC lines that wouldbe as safe as possible and thus applicable to cell therapyprotocols, we chose to use a commercially available mRNAreprogramming protocol for three reasons. First, the hostgenome integrity is secured by the fact that the methoddoes not involve the use of exogenous recombinant DNAconstructs. Second, the method does not cause permanentor prolonged ectopic expression of the transcription fac-tors, and third, it seems to be substantially simpler, faster,and potentially more efficient than most other technologiescurrently used.

FIG. 2. Expression of pluripotency markers in b-thal-iPSCs. (A) Flow cytometry results showing expression of SSEA-4and Oct3/4 in b-thal-iPSCs cultured for 10 passages. The median SSEA-4 and Oct3/4 fluorescence intensities weresignificantly higher than those of isotype controls (MFI values: 420 vs 0.721 and 12.4 vs 0.855, respectively). (B) Thepluripotency markers expression was confirmed by RT-PCR analysis. Shown are b-thal-iPSCs, embryonic pluripotent stemcell line HUES-9, and parental BM-MSCs. Lane C indicates no template control. (C) Heat map showing the expression ofpluripotency-associated genes in b-thal-iPSC lines and HUES-9 cell line relative to parental BM-MSCs.

GENERATION OF b-THAL-iPSCs USING mRNAs 5

We hypothesized that BM-MSCs can be reprogrammedefficiently to a pluripotency stage using a protocol withmodified mRNA molecules originally described for fibro-blasts. Both cell types have been grown in vitro as adherentcells exhibiting similar proliferative and differentiative po-tential (Haniffa et al., 2009), whereas BM-MSCs have beenshown to be reprogrammed to iPSCs by using other technol-ogies (Ohnishi et al., 2012). BM samples were available fromautologous backup BM grafts harvested from thalassemicpatients who were candidates for allogeneic BM transplanta-tion in our stem cell transplant unit. Our results show thatiPSC colonies were generated at day 15 after transfection ofBM-MSCs with a reprogramming efficiency of 0.05%, whichis about 74 times lower than the control cells (BJ fibroblasts).

Contradictory results have been reported on the repro-gramming efficiency of mesenchymal cells using viraltransfection methods, with ranges varying between 5% and0.0002% (de Carvalho Rodrigues et al., 2012; Ohnishi et al.,2012). However, a recent report on reprogramming of adi-pose tissue–derived MSCs by a feeder-free mRNA methodshowed a reprogramming efficiency of 0.005% (Heng et al.,2013). We demonstrated that all b-thal-iPSC lines are plu-ripotent because they express markers of pluripotency, candifferentiate in vitro to EBs, and form teratomas comprisingtissues derived from all three germ layers when injected intoimmunodeficient mice. In addition, they do not exhibit ge-netic alterations after 10 culture passages in a Matrigel-based feeder-free medium as shown by aCGH, and theyremain stable in maintaining pluripotency.

Both embryonic and iPSCs represent ideal cell popula-tions for in situ correction of the disease-causing mutationsby gene targeting methods. Using gene targeting mediatedby homologous recombination or the recent developed tran-scription activator-like effector nucleases (TALEN) tech-nology, gene-corrected iPSC lines have been generated (Maet al., 2013; Papapetrou et al., 2011; Wang et al., 2012). He-moglobin analysis of erythroblasts derived from correctediPSC lines showed restoration of the expression of full-lengthb-globin. When hematopoietic progenitors, generated fromgenetically corrected b-thal-iPSCs by homologous recombi-nation, were given to sublethally irradiated mice, an improvedhemoglobin production was observed compared with infusionof uncorrected cells (Wang et al., 2012). However, furtherstudies are needed to examine whether hematopoietic stemcells differentiated from corrected iPSCs can restore hema-topoiesis in vivo.

The differentiation potential of human iPSC lines into he-matopoietic cells has been well studied, and there are manyreports showing successful hematopoietic specific differenti-ation (Choi et al., 2009; Lengerke et al., 2009; Ye et al.,2009b). We could generate CD34 + progenitors from iPSCscultured for 10 passages by using a well-established feeder-free differentiation culture protocol (Ng et al., 2008); how-ever, most of them were CD45dim and demonstrated decreasedhematopoietic colony-forming capability. Our findings are inline with a recent report demonstrating that hemangioblasticderivatives from human iPSC lines exhibit substantially re-duced hematopoietic colony-forming capability in comparison

FIG. 3. Genetic stability and differentiation potential of b-thal-iPSCs. (A) b-thal-iPSCs cultured for 10 passages aregenetically stable as shown by aCGH. (B) In vitro differentiation of b-thal-iPSCs forming EBs in low-attachment plates.Magnification, 10 · . (C) In vivo differentiation of b-thal-iPSCs. Histology of Hematoxylin & Eosin–stained teratomasindicating tissues of all three germ layers derivation—intestinal epithelium (endoderm), cartilage (mesoderm), and neu-roepithelium (ectoderm). Magnification, 20 · .

6 VARELA ET AL.

to those derived from human ESC lines (Feng et al., 2010).The decreased hematopoietic differentiation capacity has beenalso observed and reported by others investigators in early-passage iPSCs originated from somatic cells other than he-matopoietic cells (Kim et al., 2011; Polo et al., 2010).

Recent studies showed that during the initial culturepassages iPSCs maintain a set of epigenetic and transcrip-tional markers of their somatic cell of origin, a phenomenondefined as epigenetic donor cell memory. Such a residualtranscriptional and epigenetic memory may be in favor of anin vitro differentiation potential of iPSCs back into theirtissue of origin rather than into other cell types (Bar-Nuret al., 2011; Kim et al., 2010). Remarkably, this tendency ofearly-passage iPSCs to differentiate toward the cell lineageof origin seems to be lost over time after extending culturepassaging ( > 25 passages) (Vitaloni et al., 2014).

One major hurdle for efficient generation of iPSC-derivedhematopoietic stem cells is that all differentiation proto-cols achieve hematopoietic differentiation, which is limited tothe generation of CD34+ hematopoietic stem/progenitor cellswithout engraftment potential and mature cells. Moreover,these CD34+ cells exhibit limited growth and expansioncapability, and hence are not suitable for any in vivo use.Nevertheless, the generation of hematopoietic stem cells orprogenitors with a limited proliferative potential may not bean unsolved problem, because it appears that the in vitro self-

renewal potential can be activated in differentiated cellswithout tumorigenic transformation by combined ectopicoverexpression of cMyc and Klf4 (Aziz et al., 2009). Moreimportantly, Doulatov et al. reported recently a new approachthat combines directed differentiation with transcription-based reprogramming to convert lineage-restricted CD34+

CD45+ myeloid precursors derived from human pluripotentstem cells into hematopoietic multilineage progenitors thatcan be expanded in vitro and engrafted in vivo (Doulatovet al., 2013). A detailed in vitro and in vivo functionalcharacterization of the iPSC-derived CD34+ hematopoieticstem/progenitor cells, including comparison with their normalBM counterparts so as to demonstrate their repopulatingpotential, must be an absolute requirement for each iPSC linebefore any possible clinical use.

In this study we have shown that reprogramming of BM-MSCs to pluripotent stem cells is feasible by applying thesame synthetic mRNA protocol originally optimized for fi-broblasts. Using the above methodology, we were able togenerate three unequivocally safe b-thal-iPSC lines suitablefor use in regenerative medicine.

Acknowledgments

We are grateful to Miltenyi Biotec, Bergisch Gladbach,Germany, for providing the reprogramming kit and continued

FIG. 4. In vitro hematopoietic differentiation of b-thal-iPSCs. (A) (Left) A round area consisting of single small non-adherent cells on day 13 of culture in STEMdiff APEL Medium supplemented with growth factors. Magnification, 10 · .(Right) May Gruenwald–Giemsa-stained cytocentrifuge preparations after 13 days of differentiation. Magnification, 100 · .Morphology is compatible with those of immature hematopoietic cells. (B) Flow cytometry analysis of single cells obtainedfrom day 13, demonstrating a cell population (10.2%) that is CD34 + (image b) but CD45dim (image c). Image a indicates noexpression of CD34 antigen on b-thal-iPSCs initiating the differentiation cultures.

GENERATION OF b-THAL-iPSCs USING mRNAs 7

support in our research program. We also thank Dr. K. Ste-fanaki for the histopathological assessment of teratoma tissue.

Author Disclosure Statement

The authors declare that no conflicting financial interestsexist.

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8 VARELA ET AL.

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Address correspondence to:Ioanna Varela, PhD

Stem Cell Transplant UnitAghia Sophia Children’s Hospital

Thivon and Levadias11527 Athens, Greece

E-mail: ioanvarela@gmail.com

GENERATION OF b-THAL-iPSCs USING mRNAs 9