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TISSUE-SPECIFIC STEM CELLS

Thymidine Analogs Are Transferred from Prelabeled Donor to Host Cells in the Central Nervous System After Transplantation: A Word of Caution

^a Stem Cell Institute,
^b Department of Neurosurgery,
^c Graduate Program in Neuroscience, University of Minnesota, Minneapolis, Minnesota, USA;
^d Clínica Universitaria, Universidad de Navarra, Pamplona, Spain;
^e Department of Cell Biology, Instituto Cavanilles and Centro de Investigacion Principe Felipe, University of Valencia, Valencia, Spain;
^f Department of Neuroscience, University of Minnesota, Minneapolis, Minnesota, USA;
^g Division of Neurosciences, Centre for Applied Medical Research, Pamplona, Spain

Key Words. Adult bone marrow stem cells • Label • Bromodeoxyuridine • Thymidine analog • Control • Transplantation • Neural differentiation • In vivo tracking

Correspondence: Catherine M. Verfaillie, M.D., Stem Cell Institute, Department of Medicine, University of Minnesota, 420 Delaware Street, Minneapolis, Minnesota 55455, USA. Telephone: 612-625-0602; Fax: 612-624-2436; e-mail: verfa001{at}umn.edu

Received on September 22, 2005; accepted for publication on December 5, 2005.

	ABSTRACT

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Thymidine analogs, including bromodeoxyuridine, chlorodeoxyuridine, iododeoxyuridine, and tritiated thymidine, label dividing cells by incorporating into DNA during S phase of cell division and are widely employed to identify cells transplanted into the central nervous system. However, the potential for transfer of thymidine analogs from grafted cells to dividing host cells has not been thoroughly tested. We here demonstrate that graft-derived thymidine analogs can become incorporated into host neural precursors and glia. Large numbers of labeled neurons and glia were found 3–12 weeks after transplantation of thymidine analog-labeled live stem cells, suggesting differentiation of grafted cells. Remarkably, however, similar results were obtained after transplantation of dead cells or labeled fibroblasts. Our findings reveal for the first time that thymidine analog labeling may not be a reliable means of identifying transplanted cells, particularly in highly proliferative environments such as the developing, neurogenic, or injured brain.

	INTRODUCTION

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Thymidine analogs have been used to label dividing cells in the central nervous system (CNS) for several decades [1, 2]. Thymidine analogs have also been used extensively to label cells prior to transplantation, thus allowing the fate of cells transplanted into the CNS to be determined [1, 3, 4]. As such, thymidine analogs have been used as a label to follow the migration and differentiation of stem cells, including neural stem cells [5, 6] and embryonic stem cells [7, 8], into neurons and glia after transplantation into the developing [8] and adult [4] brain.

During the past 6 years, several studies have indicated that marrow stromal cells or mesenchymal stem cells (MSCs) grafted into the brain of mice and rats can also differentiate in a region-specific manner into neurons and/or glia, suggesting greater lineage differentiation ability than was previously thought possible [9–12]. We have also shown that multipotent adult progenitor cells (MAPCs), another stem cell population isolated from bone marrow, can contribute to the brain of mice in a region-specific manner when introduced in the blastocyst [13, 14]. We wished to determine whether MAPCs could also engraft and differentiate in the brains of postnatal animals.

	MATERIALS AND METHODS

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Cell Isolation Labeling and Preparation
MAPCs [13] and fibroblasts were isolated from mice expressing enhanced green fluorescent protein (GFP) under the ß-actin promoter [15], ROSA26 mice (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org), and Sprague-Dawley rats (Harlan, Indianapolis, IN, http://www.harlan.com). Rat MAPCs were transfected with lentivirus-GFP, and stable expression was demonstrated for >35 passages. Ten micromolar bromodeoxyuridine (BrdU) or chlorodeoxyuridine (CldU) was added to the culture media 2 days prior to transplantation. Cells were washed, trypsinized, centrifuged, and rewashed prior to transplantation or in vitro coculture.

Neonatal Transplantation
Animal studies were performed in accordance with guidelines set forth by the Institutional Animal Care and Use Committee at the University of Minnesota. Two microliters containing approximately 100,000 cells were stereotaxically transplanted into the brain of P5 neonatal animals as described [16]. Briefly, animals were anesthetized by cooling under 10 cm of wet ice for 1 minute per gram, then fixed in a Kopf hypothermic neonatal frame (David Kopf Instruments, Tujunga, CA, http://www.kopfinstruments.com). Cells were injected using a 26-gauge needle attached to a 10-µl Hamilton syringe (Hamilton, Reno, NV, http://www.hamiltoncompany.com). Coordinates (in mm) were as follows: i.c.v., +1.0 anteroposterior (AP), ±1.2 mediolaternal (ML), –2.1 dorsoventral (DV) from bregma (n = 35 in multiple experiments including live and dead LacZ and GFP MAPCs); cerebellum, –1.0 AP, ±1 ML, –3.0 DV from lambda (n = 2 LacZ MAPCs).

Transuterine Transplantation
Two microliters containing approximately 50,000 BrdU-labeled fibroblasts were injected into the lateral brain ventricles of embryos (E14.5) of one pregnant CD-1 mouse using a Harvard programmable injector (Harvard Apparatus, Holliston, MA, http://www.harvardapparatus.com) and flexible 30-gauge Hamilton needle.

Transplantation into the Subventricular Zone and Rostral Migratory Stream
Dead fibroblasts (150,000 in 2 µl) were transplanted into the subventricular zone (SVZ) (right hemisphere) and rostral migratory stream (RMS) (left hemisphere) of three adult (7–8 weeks old) CD-1 mice (six grafts total) using the following coordinates: RMS: +2.2 AP, ±1.1 ML, –3.2 DV; SVZ: +1.3 AP, ±1.2 ML, –3.0 DV (mm relative to bregma). The olfactory bulb ipsilateral to each graft was analyzed at 3 weeks to assess the presence of graft-derived BrdU in newly born olfactory bulb neurons.

Transplantation into Ischemic Rat Brain
Permanent middle cerebral artery occlusion was performed in two 180–220 g spontaneously hypertensive rats. BrdU-labeled GFP fibroblasts (75,000 in 3 µl) were stereotaxically transplanted into the surrounding penumbra at each of three locations. The coordinates used were as follows: 1) 1.0 AP, 2.0 ML, –1.2 DV; 2) –3.0 AP, 1.5 ML, –1.2 DV; 3) –6.0AP, 2.0 ML, –1.2 DV [17]. Animals were sacrificed 7 days after transplantation.

Striatal Transplantation of ³H-T–Labeled Cells
GFP rat multipotent adult progenitor cells (rMAPCs) were cultured in the presence of 0.20 mCi/ml of tritiated thymidine (TRA120; GE Healthcare Life Sciences–Amersham Biosciences, Little Chalfont, Buckinghamshire, U.K., http://www.amershambiosciences.com) for 20 hours prior to cell preparation. Twenty-two normal adult Sprague-Dawley female rats (250 g) were anaesthetized, and 200,000 cells in 8 µl were injected unilaterally into striatum using coordinates – 0.2 AP, +2.6 ML, and –5.4 DV from bregma.

Controls
To control for the possibility that thymidine analog from transplanted cells may be incorporated into dividing host cells, cells were prepared as described but freeze-thawed three to four times on dry ice or liquid nitrogen immediately before transplantation. Absence of cell viability was confirmed by trypan blue inclusion and by replating in cell culture. Supernatant from transplanted cells (n = 8) was removed following the final centrifugation during cell preparation and injected as a negative control for labeled cells. Cells and supernatant were injected in parallel using the following coordinates: i.c.v., 0.0 AP, +1.0 ML, –2.1 DV or +1.0 AP, ±1.0 ML, –2.1 DV (from bregma).

In Vitro Transfer
Eight hundred thousand tritiated thymidine (³H-T)-labeled rMAPCs were cocultured in 12-well cell culture inserts (0.4 µm pore size) for 2 days to determine whether unlabeled cells could take up ³H-T released from labeled cells in vitro. Three variations of ³H-T–labeled rMAPCs were used for coculture: live cells, cells killed by repeated freeze-thaw (liquid nitrogen), and dead cells subsequently treated with DNase (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) for 30 minutes at room temperature before coculture. Growth medium alone and growth medium with 0.20 µCi/ml ³H-T were used as negative and positive controls for ³H-T uptake, respectively. Cells were fixed and processed for thymidine autoradiography after 2 days. Similar results were obtained using BrdU-labeled fibroblasts (data not shown).

Immunohistochemistry
Animals were transcardially perfused with phosphate-buffered saline followed by 4% formaldehyde. Brains were postfixed for 24 hours and then transferred to 30% sucrose until they sank. Forty- to fifty-micrometer floating sections were processed using BrdU pretreatment as described [4]. Primary antibodies were GFP (1:1,000; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com); neuron-specific nuclear protein (NeuN) (1:100; Chemicon, Temecula, CA, http://www.chemicon.com); nestin 401 (1:50; Chemicon); BrdU (1:250; Accurate; Accurate Chemical and Scientific, Westbury, NY, http://www.accuratechemical.com); BrdU (1:50; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), which recognized iododeoxyuridine (IdU) for double labeling of CldU with IdU [18]; glial fibrillary acidic protein (1:500; DAKO, Glostrup, Denmark, http://www.dako.com); and 2',3'-cyclic nucleotide 3'-phosphodiesterase (1:500; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Secondary antibodies were coupled to Cy3, Cy5, fluorescein isothiocyanate (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com), Alexa 488, Alexa 555, and Alexa 647 (Molecular Probes). The Tyramide Signal Amplification Kit (Molecular Probes) was used according to the manufacturer’s directions for identification of GFP in animals injected with GFP-rMAPCs. Images were obtained via fluorescence and confocal microscopy and processed using Adobe Photoshop. Confocal single optical sections were approximately 0.4 µm.

Electron Microscopy and [Methyl-³H]-Thymidine Autoradiography
Sagittal 200-mm sections were cut on a Vibratome tissue slicer (Leica Microsystems Nussloch GmbH, Heidelberg, Germany, http://www.leica-microsystems.com). The sections were postfixed in 2% osmium for 2 hours, rinsed, dehydrated, and embedded in Araldite (Durcupan; Fluka BioChemika, Sigma-Aldrich). Serial 1.5-µm-thick semithin sections were cut with a knife and mounted onto slides, dipped in autoradiographic emulsion (Kodak NTB2), exposed for 4 weeks at 4°C, developed in Kodak D-19, and counterstained with 1% toluidine blue. A cell was considered labeled if 10 or more silver grains overlaid the nucleus and the same cell was labeled in three adjacent sections. Seventy ³H-T-labeled cells identified in the semithin sections were selected for electron microscopic examination. Semithin sections were glued (Super Glue) to Araldite blocks and detached from the glass slide by repeated freezing (in liquid nitrogen) and thawing. The block with the flat semithin section was mounted in the ultramicrotome. Ultrathin sections were cut with a diamond knife and examined under a Jeol JEM 1010 electron microscope to determine which cell types incorporated ³H-T [19].

Cell Counting and Image Analysis
³H-T-Labeled cells were counted in representative semithin sections along the mediolateral axis in areas of 100 µm wide, up to 700 µm from the centre of the graft. Consistently in all the sections, width of the graft area was approximately 200 µm in total. Eight hundred four and six hundred sixty-three ³H-T cells were counted in representative slices at 48 hours and 7 days postinjection, respectively. A cell was considered labeled if 10 or more silver grains overlaid the nucleus in three adjacent sections. Quantification of the percentage of GFP-labeled cells at various distances from the graft area was achieved via image analysis of GFP fluorescence using analySIS FIVE image analysis software (Olympus Europa GmbH, Hamburg, Germany, http://www.olympus.com). Percentages were calculated referring to the total area of green fluorescence in each slice. Representative sections from three different animals of each group (48 hours and 7 days) were studied.

Quantitative Polymerase Chain Reaction
Analysis for the presence of graft transgene in host brain was performed using quantitative polymerase chain reaction (PCR) [13]. Briefly, quantitative PCR was carried out on genomic DNA in Taqman SYBR green universal mix PCR buffer using an ABI PRISM 770 (PerkinElmer Life Sciences, Boston, http://www.perkinelmer.com). Primer sequences are available upon request.

	RESULTS

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CldU-Labeled Neurons and Glia and Neural Precursors Are Found After Neonatal Transplantation of CldU-MAPCs
We transplanted MAPCs derived from ROSA26 mice labeled with CldU into the neonatal mouse brain. One to twelve weeks after transplantation of MAPCs into the lateral ventricle, large numbers of CldU-labeled cells were observed in the ventricular area (Fig. 1A, 1B), olfactory bulb (Fig. 1C), dentate gyrus (Fig. 1D), corpus callosum, and cortical areas surrounding the needle tract (Fig. 1E, 1F). Labeled cells were found to express region-appropriate markers, with mostly NeuN+ cells present in the olfactory bulb and the granule cell layer of the dentate gyrus, glia in white matter tracts, and nestin-positive cells in the SVZ. One week after transplantation, numerous CldU+ cells were visible within the RMS (supplemental online Fig. 1). Labeled cells present in the olfactory bulb did not express NeuN 1 week after transplantation (supplemental online Fig. 1) but did after week 3, consistent with progressive migration and differentiation of the CldU-labeled cells. Transplantation of MAPCs into the neonatal cerebellum also resulted in numerous labeled granule, but not Purkinje, cells in parallel with normal neonatal neurogenesis of cerebellar granule cells (supplemental online Fig. 2). Continued proliferation of CldU-labeled cells was evidenced by colabeling with Ki67 at 7 days, and IdU [18] for up 12 weeks after transplantation (supplemental online Fig. 3).

View larger version (88K): [in this window] [in a new window]   Figure 1. CldU-labeled neurons, glia, and neural precursors were found after neonatal transplantation of CldU-MAPCs. (A): Widespread CldU labeling near ventricular area 12 weeks after i.c.v. injection. (B): CldU-labeled cells in the subventricular zone (1 week). Arrow: CldU-labeled cell co-expressing GFAP and nestin. (C): CldU-labeled cells in the olfactory bulb (5 weeks). Arrows: colocalization of CldU and NeuN. (D): CldU-labeled neurons (NeuN) and astrocytes (GFAP) in the dentate gyrus (12 weeks). Arrows: colocalization of CldU and NeuN; arrowheads: colocalization of CldU and GFAP. (E, F): CldU-labeled astrocytes and oligodendrocytes (CNPase) in cortex near needle tract (3 weeks). Arrows: colocalization of CldU and CNPase; arrowheads: co-localization of CldU and GFAP. Confocal series projections (A, C, E) and single optical section (B, D, F). Scale bars: 100 µm (A); 20 µm (B–F). Abbreviations: CldU, chlorodeoxyuridine; CNPase, 2' ,3'-cyclic nucleotide 3'-phosphodiesterase; GFAP, glial fibrillary acidic protein; NeuN, neuron-specific nuclear protein.

View larger version (88K): [in this window] [in a new window]   Figure 2. BrdU and CldU were transferred from donor to host cells in the developing brain. (A): Labeling of neurons (NeuN) in the olfactory bulb (left and right) and astrocytes (GFAP) in the corpus callosum (middle) 3 weeks after transplantation of dead CldU- or BrdU-labeled cells. (B): BrdU labeling in cortical, hippocampal, and olfactory bulb neurons in E14.5-injected mice (week 3). (C): BrdU labeling in the dentate gyrus after transplantation of BrdU-labeled cells but not supernatant. Direct infusion of 20 µM BrdU produced minimal labeling relative to transplantation of labeled cells. All panels are confocal single optical sections. Scale bar: 20 µm. Abbreviations: BrdU, bromodeoxyuridine; CldU, chlorodeoxyuridine; GFAP, glial fibrillary acidic protein; MAPC, multipotent adult progenitor cell; NeuN, neuron-specific nuclear protein.

View larger version (108K): [in this window] [in a new window]   Figure 3. Thymidine analogs were transferred from donor to host cells in the adult brain. (A–J): 3H-T–labeled GFP-rMAPCs transplanted into adult rat striatum. (A, F): GFP-rMAPCs remained confined to GA. Scale bars: 200 µm; 20 µm (inset in A); 50 µm (inset in C). (B, G): 3H-T–labeled cells were widely distributed up to and beyond 0.6 mm from GA (arrows). Scale bars: 100 µm. (C, H): number of 3H-T and GFP cells in slice surrounding graft. (D–J): Ultrastructure of 3H-T–labeled cells. Arrows in insets correspond to nuclei lettered in electron micrographs. Lower inset in E, intermediate filaments in astrocyte. Scale bars: 2 µm (D, E, I, J); 10 µm (insets); 0.2 µm (lower inset in E). (K): Astrocytosis surrounding cortical infarct. Top inset: GFP fibroblasts confined to GA; lower detail: colocalization of BrdU with astrocytes. Arrowheads indicate colocalization of BrdU with GFAP. Scale bars: 100 µm; 20 µm (insets). (L, M): BrdU labeling in RMS (L) and olfactory bulb (M) 3 weeks after adult RMS injection of dead BrdU-labeled fibroblasts. Inset: colocalization of BrdU with NeuN. Arrowheads indicate colocalization of BrdU with NeuN. Scale bars: 200 µm; 20 µm (inset). Abbreviations: BMC, donor-derived bone marrow cell; BrdU, bromodeoxyuridine; E, endothelium; GA, graft area; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; 3H-T, tritiated thymidine; IF, intermediate filament; Mg, microglia; NeuN, neuron-specific nuclear protein; rMAPC, rat multipotent adult progenitor cell.

View larger version (121K): [in this window] [in a new window]   Figure 4. Labeled cells contained sufficient 3H-T to label proliferating cells in vitro. (A, B): Negative and positive controls for autoradiography. (C–E): Previously unlabeled rMAPCs grown in noncontact coculture with 3H-T–labeled rMAPCs after noncontact coculture with labeled rMAPCs. Coculture transwells contained live rMAPCs (C), dead rMAPCs (killed via repeated freeze-thaw) (D), or dead rMAPCs treated with DNase prior to coculture (E). Results shown are 2 days after start of coculture. Scale bar = 25 µm. Abbreviations: 3H-T, tritiated thymidine; rMAPC, rat multipotent adult progenitor cell.

Although cells were washed multiple times prior to transplantation, we hypothesized that traces of extracellular CldU or BrdU remaining in the cell suspension could be responsible for the host labeling observed. We thus transplanted supernatant from cell preparation in parallel with cells (n = 8). BrdU labeling was not observed in any animals injected with supernatant (Fig. 2C), although substantial labeling was observed in all neonatal animals injected with labeled cells. To further verify this result, we directly injected 2 µl of 20 µM BrdU into two other animals; this concentration is double the in vitro BrdU concentration used to label cells for transplantation. Although some host cells were labeled via this technique, the amount of labeling was minimal compared with that obtained after injection of labeled cells (Fig. 2C).

Thymidine Analogs Are Transferred from Donor to Host Cells in the Adult Brain
We next tested whether or not transfer of thymidine analogs from donor to host cells may also occur in adult animals, where proliferation of host cells occurs at a lower rate. Forty-eight hours and 7 days after transplantation of ³H-T-labeled GFP rMAPCs into normal striatum, GFP-positive cells could be identified in the needle tract. These cells possessed characteristic MSC-like morphology by electron microscopy (Fig. 3B). No neuronal or glial markers were observed in these cells (data not shown). Only 5%–7% of the total number of GFP-positive cells were found outside of the graft area, all within 100 µm of the graft border (insets in Fig. 3A, 3F). By contrast, almost half of the total number of ³H-T–labeled cells present in any given graft-containing slice were found outside of the graft area, at distances of up to 600 µm and beyond (Fig. 3C, 3H).

Ultrastructural analysis of individual ³H-T–labeled cells revealed labeled astrocytes and microglia (Fig. 3E, 3I), consistent with their proliferative behavior in regions of injury [20]. ³H-T–labeled endothelium was also found in 1 out of 8 animals analyzed (Fig. 3J). Consistent with thymidine analog labeling of glia in an area of injury, BrdU-labeled astrocytes were found after injection of BrdU-labeled GFP fibroblasts into the penumbra surrounding an infarct (Fig. 3K).

To determine whether adult-born neurons may also incorporate thymidine analogs, dead BrdU-labeled fibroblasts were injected into the SVZ or RMS of adult mice. Substantial BrdU labeling of NeuN+ neurons in the olfactory bulb was observed after each transplant (n = 6; Fig. 3M), suggesting that dividing host cells may be capable of incorporating graft-derived thymidine analog label regardless of the age of the host animal, the cell type grafted, or the thymidine analog used.

Labeled Cells Contain Sufficient ³H-T to Label Proliferating Cells In Vitro
Finally, to further demonstrate that the quantity of thymidine analog present within a suspension of labeled cells is sufficient to label dividing cells, ³H-T-labeled cells (rMAPCs) were cocultured in vitro in transwells above unlabeled cells. A slight amount of labeling was observed in cells under transwells containing live labeled cells (Fig. 4C), with somewhat more labeling visible after coculture with dead cells (Fig. 4D). Overwhelming labeling, however, was observed if the dead cell lysate was first treated with DNase (Fig. 4E), suggesting that transfer of thymidine analogs from transplanted cells to host cells in vivo may be dependent upon endogenous DNase activity.

	DISCUSSION

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Although BrdU and tritiated thymidine have been used to track transplanted cells for many years [3–12, 21–25], false labeling of host neural cells has, to our knowledge, not been reported previously. It is well documented that more than 90% of cells transplanted into the CNS may die shortly after transplantation, which remains a significant limitation for neural transplantation. Consequently, there is potentially a large quantity of thymidine analog from cells progressively dying in the graft that may be processed via endogenous DNase and recycled for incorporation into dividing host cells. Many studies employing thymidine analogs as a marker of transplanted cells in the CNS do not include the controls described in this paper, such as independent confirmation of graft identity using multiple independent tracers or transplantation of dead cells or fibroblasts. However, among those that have included controls [3, 6, 8], this is the first report to our knowledge of the potential for thymidine analogs to be taken up by endogenous neural cells.

Recent studies in which nonneural cells were transplanted into the CNS have suggested that marrow stromal cells or mesenchymal stem cells may possess significantly greater plasticity than was previously realized, with the potential to differentiate into tissues from unrelated embryonic germ layers. In these studies, BrdU has been widely used as a sole marker of adult stem cells to assess their potential to integrate and differentiate in the developing or injured CNS [9–11].

Thymidine analogs are also routinely used to label dividing endogenous cells [26]. The most common fate of newly born cells in the adult brain is apoptosis [27, 28]. Whether BrdU from such apoptotic cells may again be released and thereby become available for incorporation into adjacent dividing cells has not been tested but could compromise the temporal resolution of birth date identification after single bolus injection of BrdU. Some authors have suggested that BrdU may label cells undergoing DNA repair as well as dividing cells [29], especially in the context of injury and prolonged thymidine analog administration. In the present study, however, labeling was observed in host cells undergoing normal development, as evidenced by continued proliferation and time-dependent migration and adoption of neuronal identity (supplemental online Figs. 1, 3).

	CONCLUSION

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In conclusion, we report here for the first time that thymidine analogs from transplanted cells are released in vivo and become incorporated into dividing host neural cells. We suggest that interpretation of BrdU-, CldU-, or tritiated thymidine-labeled cell transplantation studies must be performed with the utmost care and that rigorous controls should be employed in the transplantation of thymidine analog-labeled cells. We suggest that, at a minimum, parallel transplantation of dead cells or labeled fibroblasts and independent confirmation of donor identity [5, 8] should be considered before drawing conclusions about the in vivo behavior of thymidine analog-labeled cells after transplantation.

	ACKNOWLEDGMENTS

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We thank Enrique Andreu, Mark Blackstad, Dominic Schomberg, and Zhenhong Nan for technical assistance. We also thank Íñigo Narvaiza who provided the Lentivirus-GFP and I. Weissman who provided the GFP mice used in this project. Support for this project was provided by an NIH Medical Scientist Training Program grant (T.C.B., X.R.O.-G., C.D.K.), 3M (T.C.B.), University of Minnesota Biomedical Engineering Institute (T.C.B.), NIH National Research Service Award (X.R.O.-G., C.D.K.), Turner Family Fund (W.C.L.), Isaksen Fund (W.L.), Cornford Fund (W.C.L.), Cavin Fund (W.C.L.), Miller Family Fund (W.C.L.), Michael Charles Winery Fund (W.C.L.), NIH grants (C.M.V., W.C.L.), and grants from Fondo de Investigaciones Sanitarias, Gobierno de Navarra-Salud, and Fundación de Investigación Médica Mutua Madrileña del Automóvil. This work was also funded through the Unión Temporal de Empresas Project Centro de Investigación Médica Aplicada. T.C.B., X.R.O.-G., and M.G.-P. contributed equally to this work.

	DISCLOSURES

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The authors indicate no potential conflicts of interest.

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