and Dose-Related Effects on MSC Engraftment ... - Wiley Online Library

TRANSLATIONAL AND CLINICAL RESEARCH Age- and Dose-Related Effects on MSC Engraftment Levels and Anatomical Distribution in the Central Nervous Systems of Nonhuman Primates: Identification of Novel MSC Subpopulations That Respond to Guidance Cues in Brain IRYNA A. ISAKOVA,a KATE BAKER,b MARIA DUTREIL,a JASON DUFOUR,b DINA GAUPP,a DONALD G. PHINNEYa a

Center for Gene Therapy, Tulane University Health Science Center, New Orleans, Louisiana, USA; bDepartment of Veterinary Medicine, Tulane National Primate Research Center, Covington, Louisiana, USA Key Words. Mesenchymal stem cells • Rhesus macaques • Real-time polymerase chain reaction • Guidance receptors • Chemotaxis Neurodegenerative diseases

ABSTRACT Mesenchymal stem cells (MSCs) have demonstrated efficacy as therapeutic vectors in rodent models of neurological diseases, but few studies have evaluated their safety and efficacy in a relevant large animal model. Previously, we reported that MSCs transplanted to the central nervous systems (CNS) of adult rhesus macaques engrafted at low levels without adversely affecting animal health, behavior, or motor function. Herein, we injected MSCs intracranially into 10 healthy infant macaques and quantified their engraftment levels and mapped their anatomical distribution in brain by real-time polymerase chain reaction using an sry gene-specific probe. These analyses revealed that MSC engraftment levels in brain were on average 18-fold higher with a maximal observed difference of 180-fold in neonates as compared with that reported previously for young adult macaques. Moreover, engraftment levels were 30-fold

higher after injection of a low versus high cell dose and engrafted MSCs were nonrandomly distributed throughout the infant brain and localized to specific anatomical regions. Identification of unique subpopulations of macaque and human MSCs that express receptor proteins known to regulate tangential migration of interneurons may explain their migration patterns in brain. Extensive monitoring of infant transplant recipients using a battery of age appropriate tests found no evidence of any long-term adverse effects on the health or social, behavioral, cognitive, or motor abilities of animals up to 6 months post-transplant. Therefore, direct intracranial injection represents a safe means to deliver therapeutic levels of MSCs to the CNS. Moreover, expressed guidance receptors on MSC subpopulations may regulate migration of cells in the host brain. STEM CELLS 2007;25: 3261–3270

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Bone marrow derived mesenchymal stem cells (MSCs) are best characterized by their ability to differentiate into various connective tissue cell lineages [1, 2] and produce cytokines, chemokines, and adhesion molecules that regulate hematopoiesis [3–5]. MSCs have also been shown to modulate immune cell function [6 – 8] and suppress T-cell immunoreactivity in response to alloantigens [9, 10]. Accordingly, MSCs have been used successfully to treat osteogenesis imperfecta [11] and graft versus host disease [12] and to speed recovery from bone marrow transplantation in humans [13]. Previously, we showed that mesenchymal stem cells injected intracranially into newborn mice exhibit widespread engraftment throughout the brain and adopt characteristics of neural cells [14, 15]. Based on this and other studies, MSCs have been exploited to treat a variety of neurological disorders including Parkinson disease [16, 17], stroke [18 –20], and traumatic brain injury [21–23]. Moreover, MSCs engineered to overexpress acid sphingomyelinase or ␤-glucuronidase and

transplanted to the central nervous system (CNS) have been shown to delay the onset of neurological symptoms and improve cognitive function in mice afflicted with Niemann-Pick disease type A and B [24, 25] or MPS VII [26], respectively. Injection of unmodified MSCs into the cerebellum of mice was also shown to alleviate inflammatory responses that contribute to the pathogenesis of Niemann-Pick disease type C [27]. Most recently, MSCs harvested from pediatric patients awaiting bone marrow transplant for hematological malignancies were found to secrete appreciable amounts of the lysosomal enzymes arylsulfatase A, ␤-hexosaminidase A, and ␤-galactosidase, which were found to be taken up by fibroblasts from enzyme-deficient patients [28]. These findings, together with their ease of isolation, low immunogenicity, and low tumor forming potential, indicate that MSCs are well suited as vectors for treating neurological disorders. Nevertheless, no clinical studies have been performed to assess the safety and efficacy of direct intracranial MSC administration for a therapeutic intent. Previously, we reported that MSCs injected intracranially into young adult rhesus macaques engrafted within various anatomical structures along the brain neuraxis without produc-

Correspondence: Donald G. Phinney, Ph.D., SL-99, Center for Gene Therapy, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA. Telephone: (504) 988-7725; Fax: (504) 988-7710; e-mail: [email protected] Received July 9, 2007; accepted for publication October 2, 2007; first published online in STEM CELLS EXPRESS October 11, 2007; available online without subscription through the open access option. ©AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2007-0543

STEM CELLS 2007;25:3261–3270 www.StemCells.com

Intracranial MSC Administration in Nonhuman Primates

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ing any long-term adverse effects on animal health, behavior, postural, locomotor, and fine motor performance [29]. Although this study suggested that intracranial MSC administration was safe, overall MSC engraftment levels in brain appeared too low to be clinically relevant. Based on our recent findings in mice that the age of the transplant recipient significantly affected MSC engraftment levels in brain [30], we repeated these preclinical studies in infant macaques. Herein, we report that cell dose and the age of the transplant recipient have a significant effect on MSC engraftment levels in the nonhuman primate brain. Additionally, we show that MSCs engrafted in brain are dynamically distributed and preferentially localize to specific anatomical regions. Our discovery of MSC subpopulations that express neural adhesion and receptor proteins known to regulate neuronal cell migration in the CNS may provide one mechanism to explain this outcome. Lastly, by subjecting transplant recipients to an extensive battery of age and species-appropriate tests, we show that intracranial MSC administration and subsequent long-term cell engraftment in brain failed to produce any longterm adverse effects on the general health or the social, behavioral, cognitive, or motor abilities of infant transplant recipients up to 6 months post-transplant.

MATERIALS

AND

METHODS

Subjects Female, rhesus macaques (Macaca mulatta) were housed in standard infant cages individually and allowed social contact with each other on a regular basis. All aspects of animal care and scientific evaluation of the macaques were conducted in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of Tulane University.

Presurgical Neurobehavioral Assessments A total of 10 newborn female rhesus macaques were hand-reared in a nursery setting and at 7 and 14 days after birth subjected to a 20-minute battery of tests to document baseline motor function, temperament, and interactive skills using a test adapted for nonhuman primates from the Brazelton Neonatal Behavioral Assessment Scales used with humans [31]. The test items included visual orientation and attention span, state control, motor maturity, activity, reflexes and responses, fine and gross motor skills and strength, and temperamental items such as vocalization, self-quieting abilities, fearfulness, and distress (supplemental online Table 1). Test scores were grouped into four categories: orientation, control, motor maturity, and activity and compared with those obtained for 109 age-, sex-, and rearing-matched infants (M. Champoux, unpublished observation).

Isolation of MSCs MSCs were elaborated from the bone marrow of a single male rhesus macaque raised in the virus-free colony at the New England National Primate Research Center as previously described [29]. MSCs used for injections were harvested at second passage and suspended in phosphate-buffered saline (PBS) at 17 ⫻ 103 cells per microliter (low dose) or 84 ⫻ 103 cells per microliter (high dose). These doses were based on preliminary studies that revealed rhesus MSCs could be suspended at a maximal density of 85 ⫻ 103 cells per microliter without clumping or loss of viability. Moreover, to test whether the injection procedure affected MSC viability, cells were suspended at 17 ⫻ 103 or 84 ⫻ 103 cells per microliter in PBS, incubated 1 hour on ice, passed through the same syringe used for brain injections, and then cultured for several passages. Cell viability immediately after passage through the syringe and after culture in vitro for two passages was 91%, 91.6%, and 94% for the high-dose populations and 91%, 92.8%, and 95% for low-dose populations, respectively. Human MSCs were elaborated from

Table 1. Physical characteristics and experimental manipulation of the study group

Animal number

FG40 FE42 FF50 FF79 FD69 FF88 FD72 FG97 FC86 FG60

Age (weeks)

Cell dose (ⴛ106 cells)

Coordinates (AP/DV/ Lat)

End points (months)

MSC engraftment (% of injected cells)

7.5 6.0 8.7 8.0 7.0 7.9 7.0 6.5 8.0 7.0

0.5 0.5 0.5 0.5 2.5 2.5 2.5 2.5 —a —a

10/14/2–6 7/15/2–4 8/16/3–5 8/16/2–6 8/16/2–6 6/14/2–6 2/14/2–6 12/17/2–6 8/16/2–6 10/16/3–6

3 6 6 6 3 6 6 6 NA NA

0.19 12.26 4.07 9.01 1.71 0.08 0.31 0.11 NA NA

The table lists each animal’s age at the time of surgery, the cell dose administered, and the time of sacrifice after surgery. a Animals FC86 and FG60 served as sham-operated controls and received an intracranial injection of phosphate-buffered saline equal to the volume of the MSC injections. These animals were not sacrificed. Abbreviations: AP, anterior-posterior; DV, dorsal-ventral; Lat, lateral; NA, not applicable.

small volume aspirates of the iliac crest obtained from donors after informed consent as described previously [32].

Surgical Procedures Animals were immobilized with ketamine (10 mg/kg), administered buprenorphine (0.01 mg/kg), acepromazine (0.02 mg/kg), and glycopyrrolate (15g/kg), and maintained on isoflurane/O2 during the surgery. The anesthetized animals were placed in a stereotactic frame (KOPF, Tujunga, CA, http://www.kopfinstruments.com) and administered two 15-␮l injections (1.0 ␮l/minute) of male MSCs or PBS via a 25-␮l Hamilton 700 syringe with a 25-gauge needle (Hamilton Co., Reno, NV, http://www.hamiltoncompany.com) targeted to the right caudate nucleus according to coordinates determined from MRI scans of the brain (Table 1). In the latter case, animals were sedated with Telazol and then a total of 60 coronal (1 mm) and 15 sagittal images (3 mm) were obtained using a GE Signa 1.5 Tesla machine (GE Healthcare, Little Chalfont, Buckinghamshire, U.K., http://www.gehealthcare.com). The ear bars of the MRI and the surface of the brain were used as reference points. Postoperatively, animals were administered analgesics and antibiotics for 5 days. At the scheduled time points for euthanasia (3 or 6 months after surgery), animals were perfused transcardially with PBS (1 l/kg) followed by 4% paraformaldehyde (1 l/kg).

Health Monitoring All transplant recipients were subjected to routine physical examinations on a regular basis. Peripheral blood was also sampled from all transplant recipients and shams 1 week prior to surgery and at 2 weeks and 1, 2, and 3 months after surgery, and cell counts were obtained using a Hematology Analyzer Advia 120 (Bayer, Leverkusen, Germany, http://www.bayer.com). Blood samples (100 ␮l) were also stained with human antibodies that cross-react with the rhesus antigens CD4, CD8 (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), CD16, and CD20 (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) according to the manufacturer’s instructions and analyzed on a FACSCalibur System (BD Biosciences). The reactivity of peripheral blood mononuclear cells (PBMNCs) against the donor MSCs was evaluated using the Cyto-Tox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI, http://www.promega.com) according to the manufacturer’s instructions. Briefly, different numbers of PBMNCs (1 ⫻ 105, 5 ⫻ 104, or 2.5 ⫻ 104 cells) were cocultured in multiwell plates (0.32 cm2) with donor MSCs (1 ⫻ 104 cells per well) for 4 hours at 37°C and then levels of cytosolic lactate dehydrogenase released into the medium were quantified

Isakova, Baker, DuTreil et al. using a colorimetric assay. The percentage of cell-mediated toxicity was then calculated using the formula provided by the manufacturer.

Bayley Test of Infant Development All transplant recipients were assessed using the Bayley test of infant development originally developed for use with human infants but modified for nonhuman primates ranging in age from 4 months to 1 year old [33]. The 10-minute evaluation consists of problemsolving, motor, and temperament tests. Briefly, each subject is held on a table by one examiner and another places test items in front of the subject and records the responses. Twenty-one separate motor and behavioral test variables (supplemental online Table 2) were collapsed into three scores for analysis (problem solving, motor abilities, and temperament). All subjects were tested at 3 months after surgery and those not euthanized were tested again at 6 months after surgery. Individual scores were compared with an age-matched normative sample (n ⫽ 20) of infants studied at the Tulane National Primate Research Center (K. Phillippi-Falkenstein, unpublished manuscript).

Play Cage Motor and Behavior Assessments Infants were placed in a socialization/exercise cage (play cage) in groups of four to six individuals on a daily basis. Play cage motor/ behavioral activity of infants was recorded once prior to surgery at approximately 5 weeks of age and then at 1, 2, 4, and 14 weeks after surgery for all subjects and again at 20 weeks for all but the two subjects that were already sacrificed. One time point each for six individuals was not recorded due to clinical problems or insufficient elapsed time after surgery. During each 5-minute observation, 21 aspects of behavior (supplemental online Table 3) were quantified with a numerical scale in ascending order of maturity and activity [34]. These items were grouped into four categories for analysis: large motor, small motor, social, and behavioral state. A repeatedmeasures analysis of variance (ANOVA) using treatment groups and behavioral categories as independent variables and dependent variables consisting of scores at the six time points (baseline and the five post-surgical tests) was used to compare motor/behavioral activity of MSC recipients and shams before and after MSC transplantation.

Quantifying MSC Engraftment Levels in Brain by Real-Time Polymerase Chain Reaction Fixed brain tissue obtained from each transplant recipient was sliced in a brain matrix (Vibratome, St. Louis, http://www.vibratome.com) into 12–14 coronal sections of alternating thickness (3 or 6 mm). Each 3-mm brain slice was subdivided into specimens weighing 100 –200 mg, homogenized, and processed for genomic DNA extraction using a standard phenol/chloroform procedure [35]. Genomic DNA (100 ng) was then analyzed by real-time polymerase chain reaction (PCR) using primers and a TaqMan probe that target sequences in the Macaca SRY gene [29]. Real-time PCR was performed using a 7700 sequence detector (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Levels of male DNA in each sample were calculated based on a standard curve generated by serially diluting genomic DNA obtained from a naı¨ve male rhesus macaque into that obtained from a female macaque, which was also used as a negative control. All reactions were run in quadruplicate and then averaged.

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94°C for 3 minutes and a final extension step at 72°C for 10 minutes. Products were visualized by electrophoresis through a 1.5% agarose gel and sequenced to confirm their identity. The following PCR primers were used: macaca cdh2, 5⬘-TGCGCGTGAAGGTTTGCCAGTGT-3⬘ and 5⬘-GCCCCCAGTCGTTCAGGTAATCATAGTCC-3⬘; human ninj1, 5⬘-CGGCCA AGCACCACGCAGGAG-3⬘ and 5⬘-AGCCCAGGCACTTTAGACCAGAG-3⬘; macaca neo1, 5⬘-CTTGAGAACGCAGAATGAGCCAGCAGACT-3⬘ and 5⬘-GCCAACCCCAGGAACAACCCACAC-3⬘; human nrp1, 5⬘-CACCCGCACCTCATTCCTACATCA-3⬘ and 5⬘-GTTGGCCTGGTCGTCATCACATTC-3⬘; macaca nrp2, 5⬘-GAAGAGATCCACCCCCAAGCACT-3⬘ and 5⬘-CAGCCAACAGAAACCCAGCCAGAG-3⬘; human robo1, 5⬘-CTGTGCCGCCACCTGCTATAAAGTCACC-3⬘ and 5⬘CCAACAGCGAGGGGAATAGGAAGGC-3⬘.

Fluorescent-Activated Cell Sorting Human MSCs (2.5 ⫻ 105) were suspended in 50 ␮l of wash buffer (0.1% sodium azide, 1.0% BSA in PBS) containing 5 ␮g of the appropriate primary antibody (BD Pharmingen) and incubated for 20 minutes on ice. Cells were washed twice with 200 ␮l of wash buffer and where necessary were incubated for 20 minutes in wash buffer (100 ␮l) containing 5 ␮g of a fluorochrome-conjugated secondary antibody (BD Pharmingen). The extent of cell labeling was evaluated using a Beckman Coulter Model Epics XL flow cytometer (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). Isotype controls were run in parallel using the same concentration of each antibody tested.

Binding and Chemotaxis Assays Binding assays were performed using Corning EIA high-binding 96-well plates (Fisher Scientific International, Hampton, NH, http:// www.fisherscientific.com). Wells were precoated with 2–50 ␮g/ml (200 ␮l) recombinant mouse netrin 1, recombinant human cadherin 2, or bovine fibronectin (R&D Systems Inc., Minneapolis, http:// www.rndsystems.com) for 1 hour at 37°C. Nonspecific binding sites were blocked by incubating the wells with 0.5% BSA for 10 minutes at 37°C. Human MSCs (50,000 cells per well) suspended in DMEM ⫹ 0.25% BSA were added to the wells and incubated for 1 hour at 37°C in a humidified chamber with 5% CO2. Wells were then fixed and stained with 0.5% Crystal Violet in 20% methanol for 30 minutes and washed, and bound cells were lysed with 20% acetic acid. The absorbance of each well was measured at 595 nm. Cell migration assays were performed using the Cytoselect 24-Well Cell Migration and Invasion Assay (Cell Biolabs Inc., San Diego, http://www.cellbiolabs.com). Wells (8-␮m pore size) were coated with 500 ␮l of recombinant human VEGF165 (10 ␮g/ml), recombinant human repulsive guidance molecule (RGM-A, 25 ␮g/ ml), or recombinant human/feline CXCl12/SDF1 (25 ␮g/ml) (R&D Systems). Human MSCs (250,000 or 500,000 cells) suspended in ␣-MEM (serum-free) were distributed to well inserts and incubated with the assay plate for 24 hours at 37°C in a humidified chamber with 5% CO2. The inserts were then transferred to wells containing 400 ␮l of staining solution and incubated for 10 minutes at room temperature. Inserts were then washed, incubated in 200 ␮l of extraction solution for 10 minutes, and transferred to a 96-well plate, and their absorbance was read at 560 nm. All samples were run in duplicate and statistical differences were evaluated using a two-tailed Student’s t test.

RESULTS

PCR Total RNA was isolated from rhesus MSCs using the RNeasy kit (Qiagen, Hilden, Germany, http://www.qiagen.com) and converted to cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer’s instructions. Aliquots of cDNA (50 ng) were used as input in PCR reactions (50 ␮l) containing 200 nM dNTPs, 2.5 mM MgCl2, 100 picomoles of each primer pair, and 2.5 U of Taq DNA Polymerase (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). PCR reactions were amplified for 40 cycles consisting of 94°C for 30 seconds, 55°C–59°C for 45 seconds, and 72°C for 60 seconds with an initial heating step at

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Pre- and Postsurgical Evaluations of Neonatal Macaques All macaque subjects recruited for the study were healthy and physically normal according to a physical exam by a veterinarian. Neurobehavioral assessment scores for all 10 newborn female macaques recruited into our study fell within two standard deviations of the mean calculated for a normative control group, confirming that their neural development was normal prior to the start of the study (not shown). Subsequently, animals

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were administered two unilateral injections of sterile PBS or 0.5 ⫻ 106 (low dose) or 2.5 ⫻ 106 (high dose) MSCs obtained from a universal male donor into the caudate nucleus of the right brain hemisphere (Table 1). The average age of the macaques at the time of MSC transplantation was 7.59 weeks old. Routine physical examination of all experimental animals revealed several notable short-term side-effects associated with the surgical procedure. Specifically, a transient decrease in tactile sensation in the right foot of one transplant recipient (FD72) was evident between 4 and 8 weeks after surgery. Additionally, several animals (FF50, FF79, and FG97) developed swelling of the scalp near the incision site between 1.5 and 3 weeks after surgery. The swelling resolved itself after several weeks but resulted in a permanent, raised area of calcification on the scalps of subjects FG97 and FF79. This complication was observed previously and attributed to an immune reaction to the bone wax used to fill the cranial defect created for cell injection [29]. In contrast, routine monitoring failed to reveal any longterm adverse effects of the cell transplantation procedure on the general health of animals. Specifically, total blood cell counts were not statistically different (p ⬎ .25) prior to surgery and at 2 weeks and 1, 2, or 3 months after surgery within individual treatment groups or between treatment groups (Fig. 1A). Moreover, fluorescence-activated cell sorting (FACS) analysis of peripheral blood samples obtained prior to surgery or at 1 month after surgery failed to reveal any significant changes (p ⬎ .10) between treatment groups in the number of circulating CD4⫹ (Fig. 1B), CD8⫹ (Fig. 1C), and CD20⫹ (Fig. 1E) cells. The average number of CD16⫹ cells was also not significantly different between treatment groups prior to surgery (p ⬎ .79) but was significantly lower (p ⬍ .05) in the low-dose treatment group as compared with sham-operated controls at 1 month after surgery (Fig. 1D). Finally, coculture of PBMNCs harvested at 1 month after surgery from each transplant recipient or shamoperated control with donor-derived MSCs revealed no significant difference (p ⬎ .15) in cell-mediated lysis between treatment groups at any of the cell ratios evaluated (Fig. 1F). Therefore, transplantation of allogeneic MSCs into the CNS of newborn rhesus macaques failed to induce a systemic immune response.

Assessment of MSC Engraftment on Animal Development and Behavior All infant transplant recipients were administered a battery of age-appropriate behavioral tests designed for nonhuman primates to evaluate the effects of MSC engraftment in the CNS on their cognitive and motor skills, social development, and temperament [34]. As would be expected in rapidly developing infant primates, there was a significant effect of age on composite test scores (F [5, 36] ⫽ 5.52, p ⬍ .001) (Fig. 2A). However, there was no significant main effect for the treatment group (F [2, 16] ⫽ 1.8, p ⬎ .10) (Fig. 2B). Specifically, neither the two-way interaction between treatment and behavioral category nor the three-way interaction among time, treatment group, and behavioral category was significant. In addition, the neurodevelopment of all transplant recipients was evaluated after surgery using the Bayley test of infant development [33]. Only one individual in the low-dose treatment group (FE42) showed a score that was two standard deviations below the range of normal subjects (Fig. 2C). However, its motor scores in the play cage tests fell well within the range of other study subjects at all time points. Consequently, the poor motor performance of this individual observed during the Bayley test was not indicative of motor impairment but more likely an artifact of the individual testing session.

Figure 1. Effect of intracranial MSC administration on the number and composition of immune cells in peripheral blood and lack of alloimmune response. (A): The average (mean ⫾ SD) number of white cells in the peripheral blood of low-dose (n ⫽ 4), high-dose (n ⫽ 4), or sham-operated (n ⫽ 2) transplant recipients was quantified 5 weeks prior to surgery and then at various times after surgery. (B–E): The average (mean ⫾ SD) number of (B) CD4, (C) CD8, (D) CD16, and (E) CD20 cells in the peripheral blood of transplant recipients harvested at 5 weeks prior to surgery and 1 month after surgery was quantified by fluorescence-activated cell sorting analysis; ⴱ, p ⬍ .05. (F): PBMNCs harvested from each transplant recipient at 1 month after surgery were incubated at various ratios with donor MSCs and the amount of cell lysis quantified. Values were then averaged for low-dose (n ⫽ 4), high-dose (n ⫽ 4), or sham-operated (n ⫽ 2) transplant recipients and plotted as percent cytotoxicity. Abbreviations: HD, high dose; LD, low dose; mon(s), month(s); P/S, post surgery; wks, weeks.

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Figure 2. Effect of intracranial MSC administration on animal behavior, development, cognitive, and motor skills. (A–B): Transplant recipients were observed in a play cage setting at 2 weeks prior to surgery and then at various intervals after surgery. Data were grouped into four categories for analysis and analyzed via repeated-measures analysis of variance. A significant effect (p ⬍ .001) of age (A) on composite behavioral scores was detected but no effect (p ⬎ .1) of the treatment group (B) was detected (vertical bars denote 0.95 confidence intervals). (C): All transplant recipients were administered the Bayley test of infant development at 3 and/or 6 months after surgery. Scores were collapsed into three categories for analysis and compared with an age-matched normative sample (n ⫽ 20) of infants (gray boxes represent 0.95 confidence interval). Abbreviations: HD, high dose; LD, low dose.

Figure 3. Effect of cell dose and recipient age on MSC engraftment levels in brain. (A): Plotted is the total amount of male DNA detected by real-time polymerase chain reaction within the brains of each infant transplant recipient. (B): Male DNA levels were used to determine the percentage (mean ⫾ SD) of injected MSCs that survived in the central nervous systems (CNS) of low-dose and high-dose transplant recipients at 3 and 6 months post-transplant; ⴱ, p ⬍ .05. (C): Plotted is the total amount of male DNA detected in the CNS of infant versus young adult transplant recipients as a function of cell dose and time after injection. (D): Plotted is the average amount of male DNA detected in the CNS of all infant or young adult transplant recipients; ⴱ, p ⬍ .05. Abbreviations: HD, high dose; LD, low dose.

Quantifying MSC Engraftment Levels in Brain by Real-Time PCR Previously, we demonstrated that a real-time PCR assay targeting the SRY gene offered a reliable way to estimate engraftment levels and map the anatomical location of male MSCs injected into the CNS of female transplant recipients [29, 30]. Accordingly, brain tissue harvested from each transplant recipient was sliced into coronal sections of alternating thickness (3 and 6 mm), and the 3-mm slices were each subdivided into specimens of comparable size and location. Analysis of brain tissue during www.StemCells.com

dissection failed to reveal any signs of gross pathology including ectopic bone formation, tumors, etc. associated with MSC engraftment. Genomic DNA prepared from the specimens, which represented approximately one-third the total volume of each brain, was then analyzed by real-time PCR to quantify male DNA levels. This analysis revealed that male DNA levels ranged from 5,617–270,345 pg between transplant recipients, which represented up to a 48-fold difference (Table 1 and Fig. 3A). Extrapolating male DNA levels to cell number revealed that a significantly greater percentage (p ⬍ .05) of injected

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Figure 4. Anatomical distribution of MSCs engrafted in brain. (A): Schematic showing the average number of male MSCs, which ranged between 1 and 2,500 cells (colored bar), contained within equivalent brain specimens harvested from each transplant recipient. Brain specimens with overlapping engraftment between two (diagonal lines bordered in black) or more than three (hatched lines bordered in red) transplant recipients are denoted. Regions in white contained no detectable male DNA. The arrow points to the approximate region where MSCs were injected into the brain. (B): Plotted is the average (mean ⫾ SD) number of MSCs contained within different brain specimens from the same coronal slice. Significant differences in overall engraftment levels were evident among brain specimens containing between 1 and 50 (blue), 51 and 250 (neon), or 251 and 2,500 (green, yellow, red) MSCs; ⴱ, p ⬍ .05. (C): Plotted are the average (mean ⫾ SD) male DNA levels contained within each respective 3-mm coronal brain slice from all infant transplant recipients; ⴱ, p ⬍ .05; ⴱⴱ, p ⬍ .01. (D): Plotted are the average (mean ⫾ SD) male DNA levels contained within each respective 3-mm coronal brain slice from all infant transplant recipients sacrificed at 3 or 6 months after injection; ⴱ, p ⬍ .05; ⴱⴱ, p ⬍ .001. Coronal brain slices are numbered 1–13 in a rostral-to-caudal orientation. Abbreviations: GYR, green yellow red; m, months.

MSCs persisted in the CNS at 6 months post-transplant in the low-dose (5.05% ⫾ 3.0%) versus high-dose (0.17% ⫾ 0.12%) transplant recipients (Fig. 3B). This effect of cell dose was not attributed to differences in the viability of MSCs prior to injection, which was greater then 90% at the densities used for transplantation. In addition, male DNA levels in the CNS of infant recipients were significantly greater when compared with data obtained previously from young adult macaques, which were subjected to the same transplantation protocol and sacrificed at the same endpoints [29]. Specifically, all but one infant transplant recipient contained higher levels of male DNA in the brain as compared with the young adult counterpart (Fig. 3C). Overall, engraftment levels were on average 17.8-fold higher (p ⬍ .05) in infant recipients as compared with adults with a maximal observed difference of 180-fold (Fig. 3D). Therefore, transplantation of a lower dose of cells into the CNS of infant recipients significantly enhanced overall MSC engraftment levels in brain.

Anatomical Distribution of Engrafted MSCs To map the distribution of engrafted MSCs in brain, male DNA levels quantified in each brain specimen by real-time PCR were averaged from all eight transplant recipients, extrapolated to cell number, and then transposed onto a physical map of each corresponding coronal brain slice (Fig. 4A). This analysis revealed that engrafted MSCs were widely distributed throughout both brain hemispheres and that their overall distribution in brain overlapped between transplant recipients. The latter was evidenced by the fact that analogous brain specimens obtained

from different transplant recipients were found to harbor similar levels of male DNA (Fig. 4A, outlined squares). On average, the highest engraftment levels were evident in tissue specimens encompassing the somatosensory, primary motor, and auditory cortex, the caudate putamen, striatum, and hippocampus. Several lines of evidence indicated that the distribution of engrafted MSCs in brain was nonrandom. First, the number of engrafted MSCs averaged over all transplant recipients varied significantly between brain specimens from individual coronal slices (Fig. 4B). For example, significant differences existed between the average number of MSCs engrafted in different brain specimens from coronal slices 1, 3, 7, or 11 (p ⬍ .05). Second, MSC engraftment levels averaged over all recipients varied significantly between coronal brain slices (Fig. 4C). For example, male DNA levels were significantly lower in coronal slices 1, 3, and 5 as compared with slice 7 (p ⬍ .05). Male DNA levels were also significantly lower in slice 5 as compared with slice 3 (p ⬍ .05) and slice 11 (p ⬍ .008). Third, differences in engraftment levels along the brain neuraxis varied significantly as a function of time post-transplantation (Fig. 4D). For example, male DNA levels in coronal slices 1 and 3 were on average 1,556-fold (p ⬍ .05) and 4,876-fold (p ⬍ .001) higher, respectively, in animals sacrificed at 6 versus 3 months after surgery. Male DNA levels were also significantly greater at 6 months versus 3 months after surgery in coronal brain slices 7 (16.2fold, p ⬍ .05) and 11 (25.1-fold, p ⬍ .03). Collectively, these data indicate that MSCs preferentially localized to specific but overlapping anatomical regions within the brains of different transplant recipients and redistributed along the brain neuraxis as a function of time post-transplantation.

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Figure 5. Rhesus and human MSC subpopulations express neural adhesion molecules and guidance receptors. (A): Polymerase chain reaction products encoding neural adhesion molecules and guidance receptors expressed by rhesus MSCs were visualized by gel electrophoresis, cloned, and sequenced to confirm their identity. (B): Fluorescence-activated cell sorting analysis of human MSCs revealed that expression of CDH2, NEO1, NRP2, and ROBO1 was restricted to specific subpopulations. In contrast, populations uniformly expressed CD44; y-axis is relative fluorescent units. Abbreviations: CDH, cadherin; FITC, fluorescein isothiocyanate; NEO, neogenin; NINJ, ninjurin; NRP, neuropilin; ROBO, roundabout homolog.

MSCs Express Adhesion Molecules and Guidance Receptors That Regulate Tangential Migration of Interneurons in Brain The nonrandom distribution of MSCs engrafted in brain suggested that a regulated process controlled their migration. Previously, we reported that murine MSCs injected intracranially into newborn and adult mice also disseminate to similar anatomical brain regions and that this process was likely mediated by neural adhesion molecules and guidance receptors expressed by the transplanted cells [30]. PCR of cDNA prepared from rhesus MSCs revealed the cells express the neural adhesion molecules cadherin 2 (CDH2) and ninjurin 1 (NINJ1) and the guidance receptors roundabout homolog 1 (ROBO1), neogenin 1 (NEO1), neuropilin 1 (NRP1), and neuropilin 2 (NRP2) (Fig. 5A). FACS analysis confirmed that expression of CDH2, NRP2, ROBO1, and NEO1 was restricted to approximately 14.5%, 7.05%, 12.3%, and 17.5%, respectively, of cells within rhesus MSC populations (not shown). Analysis of human MSCs indicated they expressed a similar repertoire of adhesion molecules and receptors. Specifically, approximately 23.3%, 19.4%, 12.0%, and 12.9% of cells within populations were found to stain with antibodies against CDH2, NRP2, ROBO1, and NEO1, respectively (Fig. 5B). The capacity of CDH2 and NEO1 to mediate binding of human MSCs to their respective ligands was evaluated in vitro. Binding of human MSCs to plates coated with CDH2 or netrin 1 (NTRN1) could be saturated in a dose-dependent manner (Fig. 6A). Moreover, binding of human MSCs to plates coated with fibronectin (FN), which was used as a control, was significantly greater at all cell doses evaluated as compared with that measured for CDH2 and NTRN1-coated plates (p ⬍ .0006), conwww.StemCells.com

sistent with the fact that the fibronectin receptor subunits CD29 and CD49e were expressed by a greater proportion of cells (96.3% and 80.1%, respectively) as compared with CDH2 (23.3%) and NEO1 (12.9%). Addition of increasing concentrations of an RGD peptide resulted in a dose-dependent inhibition of cell binding to FN-coated plates but had no effect on binding to CDH2 or NTRN1-coated plates at all concentrations tested (not shown). In contrast, preincubation of CDH2-coated plates with 20, 50, or 100 ␮g/ml recombinant human CDH2 inhibited binding of human MSCs by 70% ⫾ 1.1%, 86.5% ⫾ 4.6%, and 93.7% ⫾ 2.0%, respectively, as compared with plates coated with CDH2 alone (p ⬍ 1 ⫻ 10⫺11) (Fig. 6B). Also, preincubating human MSCs with 2.5, 5.0, 10, or 20 ␮g/ml neutralizing antibody against NEO1, the receptor for NTRN1, inhibited cell binding to NTRN1-coated plates by 72.8% ⫾ 7.2%, 78.6% ⫾ 5.2%, 87.9% ⫾ 3.6%, and 95.1% ⫾ 0.24%, respectively, as compared with untreated cells (p ⬍ 1 ⫻ 10⫺8) (Fig. 6C). Therefore, human MSCs bind to the neural adhesion molecules CDH2 and NTRN1 via homo- and heterophilic interactions, respectively. Similar results were obtained in studies conducted using rhesus MSCs. Exposure of human MSCs to the chemokine stromal cell derived factor 1 alpha (SDF-1␣) or repulsive guidance molecule A (RGM-A) induced cell migration to a significantly greater extent as compared with medium alone (p ⬍ .0003) (Fig. 6D). In contrast, pretreatment of cells with a neutralizing anti-NEO1 antibody completely abolished cell migration in response to RGM-A, indicating that this response was mediated solely by the NEO1 receptor. Next, we evaluated the function of NRP1 and NRP2 expressed by human MSCs based on their capacity to bind specific isoforms of VEGF [36]. Exposure to VEGF165 and VEGF121 also stimulated migration of human MSCs to a


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Figure 6. Binding and migration of human MSCs in vitro. (A): Binding of human MSCs to culture dishes coated with 10 ␮g/ml of FN, CDH2, or NTRN1. The number of cells bound to FN-coated plates was significantly greater at all cell doses as compared with CDH2 or NTRN1-coated plates (p ⬍ .0006). Preincubation of CDH2-coated plates with soluble CDH2 protein or (B) preincubation of MSCs with a neutralizing anti-NEO1 antibody (C) inhibited cell binding to CHD2 or NTRN1, respectively, in a dose-dependent fashion; ⴱ, p ⬍ 1 ⫻ 10⫺8. (D): Migration of human MSCs in response to SDF-1␣ and RGMA (25 ␮g/ml) or VEGF165 and VEGF121 (10 ␮g/ml) was significantly greater compared with medium alone; ⴱ, p ⬍ 5 ⫻ 10⫺9. Cell migration in response to RGMA was abolished by pretreatment of cells with a neutralizing anti-NEO1 antibody. (E): Migration of MSCs in response to VEGF165 was partially inhibited by preincubation of cells with anti-FLK1 and/or anti-FLT1 antibodies; ⴱ, p ⬍ .001; ⴱⴱ, p ⬍ 1 ⫻ 10⫺9. (F): Migration of MSCs in response to VEGF121 was completely abolished by preincubating cells with anti-FLK1 and/or anti-FLT1 antibodies; ⴱ, p ⬍ 1 ⫻ 10⫺11; ⴱⴱ, p ⬍ 5 ⫻ 10⫺5. Abbreviations: CDH, cadherin; FN, fibronectin; NEO, neogenin; NTRN, netrin; OD, optical density; RGMA, repulsive guidance molecule A; SDF, stromal cell-derived factor; VEGF, vascular endothelial growth factor.

significantly greater extent as compared with medium alone (p ⬍ .0001) (Fig. 6A). However, cell migration in response to VEGF165 was only modestly inhibited by preincubating human MSCs with neutralizing antibodies to the VEGF receptors FLK-1 and/or FLT-1 (Fig. 6E). In contrast, this pretreatment completely abolished cell migration in response to VEGF125 (Fig. 6F). This result is consistent with the fact that NRP1 and NRP2 bind to VEGF165 but not VEGF121. Therefore, NPR1 and/or NRP2 expressed by human MSCs are functional, as well. Preliminary studies have shown that human MSCs also migrate in response to medium conditioned by Neuro2A cells, which secrete Slit2 protein (not shown). Once again, similar results were obtained using rhesus MSCs.

DISCUSSION Our study demonstrates that direct intracranial injection is a safe and effective way to deliver appreciable numbers of MSCs throughout the brain for a therapeutic intent. It also reveals important new information regarding the development of MSCs as therapeutic vectors for treating neurological diseases in a clinical setting. Specifically, our data show that MSCs exhibit significantly higher engraftment levels when transplanted into the CNS of infant versus young adult macaques. Previously, we reported a similar effect of age on MSC engraftment levels in the rodent brain [30]. Although the unique attributes of the infant brain that promote enhanced MSC engraftment remain indeterminate, one explanation may be that several growth factors known to be expressed in the developing CNS, such as

FGF2 [37], also promote survival and stimulate proliferation of MSCs [38 – 40]. Our data also show that a significantly higher percentage of injected MSCs survived within the infant brain when delivered via a low dose (0.5 ⫻ 106 cells). This outcome was not due to differences in viability between the injected cell doses. Therefore, the decreased survival of MSCs injected at a high dose may result from aggregation of the cells within the brain, which may induce apoptosis/necrosis of the cells and inhibit their binding and migration in response to the appropriate ligands in the brain. Nevertheless, these outcomes are highly favorable with regard to using the cells for treating neurological sequelae associated with lysosomal storage diseases since those that result in CNS pathology have an early age of onset in humans. Therefore, it is well accepted that early intervention is necessary for any form of therapy to be effective. Accordingly, by optimizing the cell dose and injecting cells into patients at an early age, it is likely that therapeutic levels of MSCs can be achieved in the brain for treating such disorders. Our study also provides novel information about how MSCs interact with the brain microenvironment. For example, our mapping studies provide the first statistical evidence that engrafted MSCs distribute nonrandomly throughout the brain. In fact, the overlap in the distribution of MSCs between different transplant recipients provides clear evidence that the cells localize to specific anatomical brain regions. The identification of unique MSC subpopulations that express neural adhesion molecules and guidance receptors reveals one mechanism that may regulate MSC survival and migration following injection into the brain and account for their unique distribution pattern. Specifically, homo- and heterophilic interactions with neural

Isakova, Baker, DuTreil et al. adhesion proteins expressed in the host brain may promote survival of transplanted MSCs by providing a substrate for their attachment. Such interactions may be critical in that extracellular matrix proteins abundant in bone, such as fibronectin and collagens, are expressed at low levels in brain and often found only in association with blood vessels [41]. MSCs that survive in brain may then migrate to specific anatomical locations in response to guidance cues expressed by the host brain. Support for the latter comes from the fact that the distribution of expressed guidance cues in brain overlap with that of engrafted MSCs. For example, striatal cells are known to secrete semaphorins 3A and 3F, which impedes entry of interneurons that express neuropilin receptors into the striatum and redirects the cells to the cortex [42]. Similarly, Slit proteins that bind to the roundabout homolog (ROBO) family of receptors [43] are also expressed in regions of the brain where MSCs show reliable engraftment, such as the rostral migratory stream [44], hippocampus, and cerebellum [45]. Lastly, our study also provides a comprehensive assessment of the safety of intracranial MSC administration in a relevant large animal model. Specifically, evaluation of transplant recipients using a battery of age- and species-appropriate tests showed no evidence that intracranial MSC administration adversely affects animal cognition, motor function, behavior, or development. These findings are significant in that these tests have proven effective in examining how exposure to cocaine [46], lead [47– 49], methyl mercury [50], prenatal maternal stress [51], composition of infant formula, and variations in nursery care [52–54] alter such behaviors in nonhuman primates. Moreover, transplant recipients were monitored throughout a large proportion of their first year of life, during which social behavior, motor skills, and cognitive abilities are rapidly developing. Therefore, the fact that infant recipients tolerated significantly higher levels of engrafted MSCs in their CNS as compared with young adults attests to the safety of the procedure. Our study also indicates that transplantation of allogeneic

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cells from a universal male donor failed to elicit an alloimmune response in female transplant recipients. These results are consistent with several other studies showing that allogeneic MSCs exhibit low immunogenicity in a transplant setting [55–57].

CONCLUSION Collectively, these studies provide important preclinical data attesting to the safety and potential efficacy of direct intracranial MSC administration for a therapeutic intent and also provide mechanistic insight into how engrafted MSCs interact with the host CNS.

ACKNOWLEDGMENTS This research was supported in part by Grants from the National Institutes of Health to D.G.P. (R01-NS39033-01A2) and the New England National Primate Research Center (P51RR0016843). Additional funding was provided by a grant from the Millennium Trust Health Excellence Foundation to D.G.P., the Louisiana Gene Therapy Research Consortium (New Orleans), and HCA, the Health Care Company (Nashville, TN). The authors thank M. Champoux and K. Phillippi-Falkenstein for permitting the use of unpublished normative rhesus macaque infant data, K. Phillipp-Falkenstein for assistance with rhesus infant testing, and Calvin Lanclos and Allan Tucker for assistance with FACS analysis.

DISCLOSURE

OF POTENTIAL OF INTEREST

CONFLICTS

The authors indicate no potential conflicts of interest.

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