From Adult Human Mesenchymal Stem on Neurogenesis Α Enha

Enhancing Effect of IL-1α on Neurogenesis from Adult Human Mesenchymal Stem Cells: Implication for Inflammatory Mediators in Regenerative Medicine This information is current as of September 26, 2021. Steven J. Greco and Pranela Rameshwar J Immunol 2007; 179:3342-3350; ; doi: 10.4049/jimmunol.179.5.3342 http://www.jimmunol.org/content/179/5/3342 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Enhancing Effect of IL-1␣ on Neurogenesis from Adult Human Mesenchymal Stem Cells: Implication for Inﬂammatory Mediators in Regenerative Medicine1

Steven J. Greco* and Pranela Rameshwar2†

Mesenchymal stem cells (MSCs) are mesoderm-derived cells, primarily resident in adult bone marrow. MSCs show lineage specificity in generating specialized cells such as stroma, fat, and cartilage. MSCs express MHC class II and function as phagocytes and APCs. Despite these immune-enhancing properties, MSCs also exert veto functions and show evidence for allogeneic transplantation. These properties, combined with ease in isolation and expansion, demonstrate MSCs as attractive candidates for tissue repair across allogeneic barriers. MSCs have also been shown to transdifferentiate in neuronal cells. We have reported expression of the neurotransmitter gene, Tac1, in MSC-derived neuronal cells, with no evidence of translation Downloaded from unless cells were stimulated with IL-1␣. This result led us to question the potential role of immune mediators in the field of stem cell therapy. Using Tac1 as an experimental model, IL-1␣ was used as a prototypical inflammatory mediator to study functions on MSC-derived neuronal cells. Undifferentiated MSCs and those induced to form neurons were studied for their response to IL-1␣ and other proinflammatory cytokines using production of the major Tac1 peptide, substance P (SP), as readout. Although IL-1␣ induced high production of SP, a similar effect was not observed for all tested cytokines. The ␣ induced SP was capable of reuptake via its high-affinity NK1R and was found to stabilize IL-1R mRNA. IL-1 also enhanced http://www.jimmunol.org/ the rate of neurogenesis, based on expression of neuronal markers and cRNA microarray analyses. The results provide evidence that inflammatory mediators need to be considered when deciding the course of MSC transplantation. The Journal of Immunology, 2007, 179: 3342–3350.

here are ongoing research studies on the therapeutic use of Mesenchymal stem cells (MSCs) are ASCs mostly found in the stem cells, including clinical trials for various diseases (1–3). bone marrow (BM), surrounding the blood vessels and trabecula T The successful application of stem cells in these and other (6). Physiologically, MSCs exhibit a “gate-keeper” function in that trials will depend on the type of transplanted stem cell, patient health, they monitor afferent and efferent immune cell migration within disease modality, and the microenvironment of the recipient tissue. Both the BM (7–8). MSCs are attractive candidates in models of regen- by guest on September 26, 2021 adult stem cells (ASCs)3 and embryonic stem cells are attractive can- erative medicine given their ease in harvesting, isolation, and ex- didates for regenerative therapies (4). However, their transition from pansion (9). MSCs appear to bypass immune rejection through “bench to bedside” has been slow, partly due to immune rejection and veto properties, thus making them candidates for allogeneic trans- tumor formation (4). ASCs tend to be tissue-specific, thus indicating plantation (10). MSCs show lineage-specific differentiation along that their normal lineage development is limited to specialized cells osteogenic, chondrogenic, and adipogenic paths (11). MSCs have within a particular tissue or organ of residence. It could be argued that also been reported to transdifferentiate into cells of ectodermal and this seeming specificity of ASCs limits their regenerative capacity as endodermal tissue (12–15). compared with embryonic stem cells. However, some ASCs are easily Disease modality and patient health are two key variables in the attainable and show reduced propensity to transform (5). success of stem cell therapies. The concept of personalized health care practice takes into account these two factors in determining the course of treatment and measuring the predicted outcome. New *Graduate School of Biomedical Sciences, University of Medicine and Dentistry of clinical procedures, such as mathematical modeling, are at the New Jersey, Newark, NJ 07103; and †Department of Medicine, University of Med- icine and Dentistry of New Jersey–New Jersey Medical School, Newark, NJ 07103 forefront of personalized medicine (16). These models evaluate the Received for publication February 28, 2007. Accepted for publication June 27, 2007. patient’s personal profile and medical history, and mathematically The costs of publication of this article were defrayed in part by the payment of page relate it to the etiology of the disease or disorder to predict the charges. This article must therefore be hereby marked advertisement in accordance appropriate treatment. A critical consideration in mathematical with 18 U.S.C. Section 1734 solely to indicate this fact. modeling of disease or injury is the surrounding microenvironment 1 This work was supported by a grant from the F. M. Kirby Foundation. This work (17). In vitro, a MSC’s microenvironment can be closely moni- was performed in partial fulfillment of a Ph.D. thesis by S.J.G. and was done at the Department of Medicine, Division of Hematology/Oncology, University of Medicine tored to favor stem cell growth and/or differentiation. In vivo, the and Dentistry of New Jersey-New Jersey Medical School (Newark, NJ). transplanted MSCs are exposed to immune cells and mediators that 2 Address correspondence and reprint requests to Dr. Pranela Rameshwar, University of could influence the cells’ behavior. Medicine and Dentistry of New Jersey-New Jersey Medical School, Medical Sciences Human MSCs (hMSCs) can generate functional neuronal Building, Room E-579, 185 South Orange Avenue, Newark, NJ 07103. E-mail address: [email protected] cells (18, 19). We have previously shown expression of a neu- 3 Abbreviations used in this paper: ASC, adult stem cell; MSC, mesenchymal stem rotransmitter gene, Tac1, following transdifferentiation, al- cell; BM, bone marrow; SP, substance P; hMSC, human MSC; DAPI, 4Ј,6-diamidino- though translation did not occur unless the cells were stimulated 2-phenylindole, dilactate; NIM, neuronal induction medium; fSP, FITC-conjugated with IL-1␣ (18). This report used this information as the basis SP; miRNA, microRNA; MHC-II, MHC class II; RA, retinoic acid. to study how production of the Tac1-encoded neurotransmitter, Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 substance P (SP), could serve as an indicator of the interaction www.jimmunol.org The Journal of Immunology 3343 between undifferentiated MSCs or MSC-derived neuronal cells and inflammatory mediators expected in the microenvironment of an injured tissue. IL-1␣ was selected to represent a prototypical inflammatory cytokine. This report shows the ability of IL-1␣ to induce the production of SP, and to enhance the expression of neurogenesis-linked genes during neuronal induction of hMSCs. The functions of the neuronal cells with regards to SP uptake were also described.

Materials and Methods Reagents and Abs

DMEM with high glucose, DMEM/F12, L-glutamine, and B-27 supplement were purchased from Invitrogen Life Technologies. FCS, all-trans retinoic acid (RA), SP, phosphatase substrate, actinomycin and Ficoll-Hypaque were purchased from Sigma-Aldrich. Defined FCS was purchased from Atlanta Biologicals. Recombinant human basic fibroblast growth factor, IL-1␤, IL-6, TNF-␣, and IL-2 were purchased from R&D Systems. 4Ј,6- diamidino-2-phenylindole, dilactate (DAPI) and BAPTA-AM were purchased from Molecular Probes. Protease inhibitor mixture was purchased from Active Motif. Biotinylated SP was purchased from Arnell Products. Downloaded from CP-96,345, an NK1-specific antagonist, was obtained from Pfizer. Recom- binant human IL-1␣ was obtained from Hoffman La Roche. Nestin and NeuN mAbs were purchased from Chemicon International. TNFRI mAb was purchased from Abcam. Goat anti-IL-6R was purchased from R&D Systems. Rabbit anti-Notch1, rabbit anti-NK1R, ␤-actin mAb, HRP-conjugated goat anti-rabbit IgG, HRP rabbit anti-goat IgG, and HRP goat anti-mouse IgG Abs were purchased from Sigma-Aldrich. Rabbit anti- http://www.jimmunol.org/ IL-1RI and rabbit anti-IL-2R were obtained from Santa Cruz Biotechnol- ogy. Alkaline phosphatase-conjugated goat anti-rabbit IgG was obtained from Kirkegaard & Perry Laboratories. Culture of hMSCs hMSCs were cultured from BM aspirates as described (10). The use of human BM aspirates followed a protocol approved by the Institutional Review Board of University of Medicine and Dentistry of New Jersey (Newark, NJ). Unfractionated BM aspirates (2 ml) were diluted in 12 ml of DMEM containing 10% FCS (D10 medium) and then transferred to vac- uum-gas plasma treated, tissue-culture Falcon 3003 petri dishes (BD Bio- by guest on September 26, 2021 sciences). Plates were incubated, and at day 3, mononuclear cells were isolated by Ficoll Hypaque density gradient and then replaced in the culture plates. Fifty percent of the medium was replaced with fresh D10 medium at weekly intervals until the adherent cells were ϳ80% confluent. After FIGURE 1. Luciferase activity of Tac1 reporter genes in uninduced four cell passages, the adherent cells were symmetric, CD14Ϫ, CD29ϩ, MSCs and neuronal cells (days 6 and 12 (D6 and D12)). A, Schematic CD44ϩ, CD34Ϫ, CD45Ϫ, SH2ϩ, prolyl-4-hydroxylaseϪ (10). diagram of Tac1 promoter inserts, TAC1–1.2 and TAC1/NO. B, TAC1– 1.2, TAC1/NO, and reporter vector alone were transfected into uninduced Neuronal induction of MSCs MSCs, and then grown in NIM for 12 days. At select time points, cells At ϳ70–80% confluence, MSCs were trypsinized and then subcultured in were examined for the vector ampicillin resistance gene by RT-PCR to 60-mm Falcon 3002 petri dishes or in Falcon 353046 6-well plates (BD confirm stable transfection. C, Luciferase activities of TAC1–1.2 and Biosciences). For Western analyses, Northern analyses, RT-PCR, and mi- TAC1/NO in transfected, uninduced MSCs and D6 and D12 neuronal cells. croarray profiling, 104 MSCs were seeded in 60-mm tissue-culture dishes. Results are presented as the mean Ϯ SD of normalized luciferase for five 3 For transfection studies, SP ELISA and SP reuptake microscopy, 10 different experiments, each performed with a different BM donor. Normal- MSCs were seeded in 6-well tissue-culture plates. All cells were allowed izations were performed by comparing the ratio of luciferase per micro- to adhere to the culture surface overnight in D10 medium. At 20% con- gram of protein in cells transfected with vector alone, and setting this value fluence, D10 medium was replaced with neuronal induction medium p Ͻ ءء p Ͻ ء (NIM), which was comprised of Ham’s DMEM/F12, 2% FCS (Sigma- to 1. , 0.05 vs uninduced MSCs transfected with TAC1–1.2; , Aldrich), B27 supplement, 20 ␮M RA, and 12.5 ng/ml basic fibroblast 0.05 vs similar cells transfected with TAC1–1.2. growth factor. Stock solution of RA was diluted in DMSO to 20 mM. The induction FCS is permissive to commitment to other cells as opposed to the retention of pluripotency. The medium was unchanged during the entire period of induction, maximum of 12 days. Concurrent studies replacing were prepared as described previously (20). For neuronal cells, at 48 h, 50% NIM after 4 days of induction yielded similar results. All experimen- Effectene was replaced with NIM, and cells were induced for 6 or 12 days. tal endpoints were performed with a maximal confluence of 70% to control Luciferase activities in 10 ␮l of extracts were quantitated using the lucif- for contact inhibition (19). erase assay system (Promega). Normalizations were performed by comparing the ratio of luciferase per microgram of protein in cells transfected Vectors with vector alone, and setting this value to 1. To detect stable expression of luciferase-reporter vectors in transfected The 5Ј flanking region of the Tac1 gene was cloned previously and ana- cells, plasmid DNA was extracted using the Qiaprep Spin Miniprep kit lyzed (20). Fig. 1A shows the regions of various inserts as a cartoon. pGL3- (Qiagen). The extracted DNA served as DNA template for amplification of TAC1–1.2: 740 bp upstream of exon 1, exon 1 and parts of intron 1; the pGL3-basic ampicillin resistance gene (Ampr) by PCR. The primers for pGL3-TAC1/NO: 740 bp upstream of exon 1. Ampr span ϩ3302/ϩ3608 of the pGL3-basic vector (Promega). PCR was Transfection and reporter gene assay performed with the following primer pairs: (forward) 5Ј-ttt atc cgc ctc cat cca-3Ј and (reverse) 5Ј-tac gga tgg cat gac agt-3Ј. The cycling profile for Experimental vectors with luciferase-reporter gene inserts were transfected Ampr (20 cycles) is: 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s with in 50% confluent MSCs using Effectene (Qiagen). At 48 h, cell-free lysates a final extension at 72°C for 10 min. PCR (10 ␮l) were separated by 3344 IL-1␣ IN MSC-DERIVED NEURONAL CELLS Downloaded from http://www.jimmunol.org/

FIGURE 2. Neuronal induction of MSCs and Tac1 expression. A, Total RNA from uninduced MSCs and day 6 and 12 (D6 and D12) neuronal cells was studied for Tac1 expression by RT-PCR. Normalizations were performed with oligonucleotides specific for GAPDH. B, Fold change in Tac1 gene expression among uninduced, D6, and D12 cells was determined by real-time RT-PCR. Results are presented as the mean Ϯ SD fold change for three different experiments, each performed with a different BM donor. Normalizations were performed by comparing the ratio of Tac1 expression by guest on September 26, 2021 p Ͻ 0.05 vs ,ء .in uninduced cells to ␤-actin, and setting this value to 1 uninduced MSCs. electrophoresis on 1.5% agarose containing ethidium bromide. Band sizes were compared with 1-kb DNA ladder (Invitrogen Life Technologies). Semiquantitative and real-time RT-PCR Total RNA (2 ␮g) was reverse transcribed, and 200 ng of cDNA was used to amplify Tac1. For real-time PCR, Tac1 was amplified with the Platinum FIGURE 3. SP production in IL-1␣-stimulated MSCs and neuronal SYBR Green qPCR SuperMix-UDG Kit (Invitrogen Life Technologies). cells. Uninduced MSCs and day 6 and 12 (D6 and D12) neuronal cells were The primers for Tac1 span ϩ60/ϩ328 (NM_003182) with the following stimulated with 10 ng/ml IL-1␣, or unstimulated, in the presence or ab- sequences: (forward) 5Ј-act gtc cgt cgc aaa atc-3Ј and (reverse) 5Ј-ggg cca sence of 10 ␮M BAPTA-AM. Following stimulation, (A) medium or (B) ctt gtt ttt caa-3Ј. PCR were normalized by amplifying the same sample of whole cell lysate was collected from both cell subsets and SP levels were cDNA with primers specific for GAPDH, (for semiquantitative PCR) or quantified by ELISA. Results are presented as the mean SP concentra- ␤-actin (for real-time PCR). The primers for GAPDH and ␤-actin span tion Ϯ SD for five different experiments, each performed with a different ϩ212/ϩ809 (NM_002046) and ϩ842/ϩ1037 (NM_001101), respectively, p Ͻ ء with the following sequences: GAPDH (forward) 5Ј-cca ccc atg gca aat tcc BM donor. , 0.05 vs unstimulated cells. atg gca-3Ј and (reverse) 5Ј-tct aga cgg cag gtc agg tcc acc-3Ј; ␤-actin (forward) 5Ј-tgc cct gag gca ctc ttc-3Ј and (reverse) 5Ј-gtg cca ggg cag tga with the following: IL-2 (10 ng/ml), IL-1␤ (10 ng/ml), IL-6 (5 ng/ml), or tct-3Ј. Real-time PCR were performed with a 7500 Real-Time PCR System TNF-␣ (100 ng/ml). Cell growth medium was collected and competitive (Applied Biosystems). The cycling profile for semiquantitative PCR for ELISA was used to quantify SP production, as previously described (21). Tac1 (40 cycles) and GAPDH (30 cycles) was: 94°C for 30 s, 55°C for Briefly, 96-well plates were coated with a complex of streptavidin-biotin- 30 s, and 72°C for 30 s with a final extension at 72°C for 10 min. The ylated SP. Equal volumes (50 ␮l) of unknown samples and optimum rabbit cycling profile for real-time PCR for Tac1 (40 cycles) and ␤-actin (40 anti-SP were added to quadruplicate wells. For intracellular SP quantifi- cycles) was: 94°C for 15 s and 60°C for 45 s. Gene expression analysis was cation, 50 ␮l of cell-free, whole cell lysates treated with 1 ␮l of protease performed using the 7500 System SDS Software (Applied Biosystems). inhibitor mixture were used as the unknown. Bound anti-SP was detected Normalizations were performed by comparing the ratio of Tac1 expression with alkaline phosphatase-conjugated goat anti-rabbit IgG and 104 phos- in uninduced or induced cells to ␤-actin, and setting this value to 1. PCR phatase substrate (Sigma-Aldrich). SP levels were calculated from a stan- were separated by electrophoresis as described above. dard curve developed with OD at 405 nm vs 12 serial dilutions of known SP concentrations. SP quantification Western analysis Uninduced and neuronally induced MSCs were stimulated for 16 h with 10 ng/ml IL-1␣. This concentration of IL-1␣ was found to be optimal for SP Whole cell or membrane extracts were prepared from neuronal cells or quantification. In additional experiments, cells were stimulated for 16 h uninduced MSCs. Cells were trypsinized and washed in PBS (pH 7.4). The Journal of Immunology 3345

FIGURE 4. SP production by MSCs and neuronal cells stimulated with other inflammatory cytokines. Media from (A) uninduced MSCs and (B) day 6 (D6) and (C) day 12 (D12) neuronal cells stimulated with IL-2, IL-1␤, IL-6, TNF-␣, or unstimulated, was collected and SP levels were quantified by ELISA. Results are presented as the mean SP concentration Ϯ SD for five different experiments, each performed with a different BM donor. D, Membrane extracts from uninduced, D6, and D12 cells stimulated with IL-1␣ or unstimulated, were prepared and analyzed by Downloaded from Western blot with anti-IL-1RI, -IL- 6R, -TNFRI, -NK-1, and -IL-2R␣ Abs. Normalizations were performed with anti-␤-actin Ab. Representative blot is shown for three different experiments, each performed with a Ͻ ء

different BM donor. , p 0.05 vs http://www.jimmunol.org/ unstimulated cells.

After this, 30 ␮lof1ϫ lysis buffer (Promega) was added and the cells were was transferred to nylon membranes (S & S Nytran) and then hybridized subjected to freeze/thaw cycles in a dry ice/ethanol bath. Cell-free, whole with IL-1RI-specific cDNA probe, randomly labeled with 3000 Ci/mmol by guest on September 26, 2021 cell lysates were obtained by centrifugation at 4000 ϫ g for 5 min at 4°C. [␣-32P]dATP (DuPont/NEN). Probes were labeled with the Prime-IT II Total protein was determined with a Bio-Rad DC protein assay kit. Extracts random primer kit (Stratagene). To normalize RNA loading, membranes (20 ␮g) were treated with 1 ␮l of protease inhibitor mixture, and analyzed were stripped and reprobed with cDNA for 18S rRNA. Hybrids were de- by Western blots using 4–20% SDS-PAGE precast gels (Bio-Rad). Pro- tected by exposures in a phosphoimager cassette (Molecular Dynamics), teins were next transferred onto polyvinylidene difluoride membranes which was scanned at different times from 6 to 24 h with the Typhoon 9410 (PerkinElmer). Membranes were incubated overnight with primary Abs Molecular Imager phosphoimager system (Molecular Dynamics). The IL- and then detected the following day by a 2-h incubation with HRP-conju- 1RI cDNA probe was prepared by RT-PCR using primers that span gated IgG. All primary and secondary Abs were used at final dilutions of ϩ1026/ϩ1373, with the following sequences: (forward) 5Ј-aga ata cac atg 1/1000 and 1/2000, respectively. HRP was developed with chemilumines- gta tag-3Ј and (reverse) 5Ј-agt cat ccc ttc cat aaa-3Ј. The RNA template for cence detection reagent (PerkinElmer). The membranes were stripped with RT-PCR was derived from MSCs induced for 16 h with 10 nM SP. Restore Stripping Buffer (Pierce) for reprobing with other Abs.

SP labeling and reuptake Biotinylated cRNA synthesis SP was labeled with FITC using the EZ-Label FITC Protein Labeling kit Total RNA was extracted from day 12 neuronal cells using the RNAque- (Pierce Biotechnology), according to manufacturer’s recommended guide- ous-4PCR kit (Ambion). The procedure followed manufacturer’s specified lines. Briefly, lyophilized SP was dissolved in borate buffer and then in- guidelines. Biotinylated-UTP cRNA probes were synthesized from the ex- cubated for1hindimethylformamide containing FITC fluorescent dye. tracted RNA using the TrueLabeling-AMP 2.0 kit from SuperArray. Excess fluorescent dye was removed in PBS buffer using a Slide-A-Lyzer Briefly, cDNA was synthesized from 1 ␮g of total RNA at 50°C for 1 h. MINI Dialysis Unit. FITC-labeled SP (fSP) was then stored at Ϫ20°C at a Next, the cDNA was incubated overnight at 37°C with RNA polymerase stock concentration of 100 mM. enzyme and 10 mM biotinylated-UTP (Roche) to synthesize labeled cRNA For reuptake studies, stock fSP was diluted in 0.1% PBS/BSA and added probes. cRNA was purified using the SuperArray Arraygrade cRNA to uninduced MSCs and day 12 neuronal cells at final concentrations of 0.1 or 1 ␮Mfor1hat37°C. To show SP specificity, day 12 cells were co- Cleanup kit. incubated with 1 ␮M fSP and 10 ␮M unlabeled SP or 0.01 ␮M CP-96,345, an NK1-specific antagonist, for 1 h. Following reuptake, cells were washed with PBS (pH 7.4) and then fixed with 3.7% formaldehyde for 5 min. Cells Microarray analysis were next counterlabeled with the nucleus-specific dye, DAPI, at a final Oligo GEArray Human Neurogenesis and Neural Stem Cell Microarrays concentration of 0.3 ␮M. Labeled cells were examined on a three-color were purchased from SuperArray. The protocol for the microarray analyses fluorescent microscope (Nikon Instruments). followed manufacturer’s recommendations. Briefly, day 12 neuronal cell Northern analysis cRNA probes were hybridized to the microarrays overnight at 60°C with gentle rotation in a hybridization oven. The following day, alkaline phos- Northern analysis for IL-1RI mRNA in neuronally induced MSCs was phatase-conjugated streptavidin was bound to the arrays for chemilumi- performed as described previously (22). Briefly, day 12 neuronal cells were nescent substrate detection by autoradiography. Array spot density and treated with 10 ␮g/␮l actinomycin and then incubated with or without 10 differential probe expression was calculated using SuperArray’s GEArray nM SP. At 4-h intervals, up to 36 h, total RNA was extracted and 10 ␮g Expression Analysis Suite software. Spot density was normalized to select of each sample was separated by electrophoresis in 1.2% agarose. RNA positive and negative controls spotted onto each array. 3346 IL-1␣ IN MSC-DERIVED NEURONAL CELLS

FIGURE 5. Tac1 expression in MSCs and neuronal cells stimulated with inﬂammatory cytokines. Total RNA from (A) uninduced MSCs and (B) day 6 (D6) and (C) day 12 (D12) neuronal cells stimulated with IL-1␣, IL-2, IL-1␤, IL-6, TNF-␣,orun- stimulated was studied for Tac1 expression by real-time RT-PCR. Re- sults are presented as the mean Ϯ SD fold change for three different experiments, each performed with a differ- Downloaded from ent BM donor. Normalizations were performed by comparing the ratio of Tac1 expression in unstimulated cells to ␤-actin, and setting this value to 1. .p Ͻ 0.05 vs unstimulated cells ,ء http://www.jimmunol.org/

Statistical analysis question, we performed semiquantitative RT-PCR for Tac1. Fig. by guest on September 26, 2021 Statistical data analyses were performed with ANOVA and Tukey-Kramer 2A shows time-dependent increase in band intensities for Tac1 multiple comparisons test. A value of p Ͻ 0.05 was considered significant. mRNA. Normalization with GAPDH showed an increase in band intensities at day 12 neuronal induction. To verify the results Results shown in Fig. 2A, we studied the mRNA using a quantitative Reporter gene activity of Tac1 fragments in uninduced MSCs method. Quantification by real-time RT-PCR showed significant and neuronal cells ( p Ͻ 0.05) increase in days 6 and 12 neuronal cells compared with uninduced MSCs (Fig. 2B). In summary, the results show in- The ideal stage of stem cell differentiation for tissue implantation, creased levels of Tac1 mRNA during the course of neuronal that is transplantation of undifferentiated or differentiated cells, is induction. largely unknown. Thus, our studies encompassed the early and late stages of neuronal induction. The first set of experiments examined ␣ the expression of Tac1 at different times of neuronal induction, SP production in IL-1 -stimulated MSCs and neuronal cells with reporter gene vectors. Because these studies required long- We assessed whether Tac1 expression correlated with production term experiments, we first determined feasibility by transfecting of the gene’s encoded peptide, SP. Uninduced MSCs and days 6 uninduced MSCs with the pGL3 luciferase reporter containing the and 12 neuronal cells were stimulated with 10 ng/ml of the in- 5Ј flanking region of Tac1: TAC1–1.2 and TAC1/NO (Fig. 1A). flammatory cytokine, IL-1␣, or were unstimulated. After 16 h, the Stability of pGL3 transfection was determined by PCR for Ampr culture medium was assayed for SP by ELISA. At all time points, using plasmid DNA extracted from cells subjected to neuronal IL-1␣ induced the production of significant ( p Ͻ 0.05) SP as com- induction. Bands were obtained up to day 12 at the predicted size pared with the unstimulated cells (Fig. 3A). of 300 bp (Fig. 1B). Reporter gene activity in uninduced MSCs and Next, we evaluated whether SP levels in IL-1␣-stimulated me- days 6 and 12 neuronal cells showed increased activity for both dium could be due to the release of intracellular SP stores or de fragments as differentiation progressed (Fig. 1C). TAC1/N0, novo production. To this end, whole cell lysates from uninduced which lacks exon 1 and the 5Ј region of intron 1, yielded signif- MSCs and day 6 and 12 neuronal cells, stimulated with IL-1␣ or icantly ( p Ͻ 0.05) greater luciferase activity in the uninduced and unstimulated, were studied. Low to undetectable SP was found in day 6 cells (Fig. 1C, s) as compared with TAC1–1.2 (Fig. 1C, Ⅺ). all unstimulated cells (Fig. 3B, Ⅺ), thus signifying that the low There was no significant ( p Ͼ 0.05) difference between TAC1-NO levels in the medium (Fig. 3A, Ⅺ) could not be due to internally and TAC1–1.2 at day 12 induction (Fig. 1C, right bars). stored SP. Low to undetectable SP was also found in all stimulated cells (Fig. 3B, s), thus signifying production and release into the Endogenous expression of Tac1 medium (Fig. 3A, s). This section determined whether the reporter gene activity (Fig. To inhibit neurotransmitter release, cells were stimulated with 1C) relates to endogenous expression of Tac1. To address this IL-1␣ or unstimulated in the presence of the calcium chelator, The Journal of Immunology 3347

FIGURE 7. IL-1␣ stimulation of MSCs promotes neuronal differentiation. Uninduced MSCs and day 6 (D6) and day 12 (D12) neuronal cells were stimulated for 16 h with increasing concentrations of IL-1␣ (1, 5, and

10 ng/ml). Whole cell extracts were prepared and analyzed by Western blot Downloaded from with (A) anti-Nestin, -NeuN, and (B) -Notch1 Abs. Normalizations were performed with anti-␤-actin Ab. Representative blots are shown for three different experiments, each performed with a different BM donor. http://www.jimmunol.org/ of calcium-mediated transmitter release. The results verify that the effects of IL-1␣ are not due to the induction of SP release from internal stores.

SP production by IL-1␤, IL-2, IL-6, and TNF-␣ This section explores the specificity of IL-1␣ in SP production. To address this question, we first studied the response in SP production to other inflammatory cytokines, IL-2, IL-1␤, IL-6, and

TNF-␣. Uninduced MSCs stimulated with IL-2 showed signifi- by guest on September 26, 2021 cantly ( p Ͻ 0.05) increased SP levels compared with unstimulated cells or cells stimulated with other cytokines (Fig. 4A). SP levels were increased in TNF-␣-stimulated day 6 neuronal cells (Fig. 4B) and day 12 cells stimulated with IL-6 or TNF-␣ (Fig. 4C). The results show a blunted response by IL-2 in day 6 and 12 neuronal cells (Fig. 4, B and C) and by IL-6 and TNF-␣ in uninduced MSCs (Fig. 4A). SP production was not observed in uninduced or induced cells stimulated with IL-1␤. We next determined whether the blunting effects were due to a lack of expression of the cytokines’ respective receptors during the course of induction. Membrane extracts were studied by Western blot using Abs specific for the cytokine receptors. The results show a light band for the IL-2R in the uninduced cells, but bright bands in the day 6 and 12 cells (Fig. 4D, row 1). In contrast, dense bands FIGURE 6. IL-1RI mRNA stabilization by internalized SP in day 12 were observed in uninduced and induced cells for IL-1RI, IL-6R, (D12) neuronal cells. A, Uninduced MSCs and D12 neuronal cells were in- and TNF-RI (Fig. 4D, rows 2–4). We next determined whether the cubated with 0.1 ␮M(top and middle, left panels)or1␮M fSP (top and SP receptor, neurokinin 1 (NK1), was expressed, because this middle, right panels), and then counterlabeled with nucleus-specific DAPI. could signify possible reuptake of SP after IL-1␣ stimulation. We SP-specific internalization in D12 cells was shown by coincubation with 10 observed dense bands for NK1 at all time points (Fig. 4D, row 5). ␮M unlabeled SP (bottom, left panel) or 0.01 ␮M SP receptor, NK1, antag- Stimulation of the cells with IL-1␣ had little to no effect on ex- onist (bottom, right panel). Figure shows representative labelings of three different experiments, each performed with a different BM donor. Images are pression of the studied receptors (Fig. 4D). shown at ϫ20 magnification. B, D12 cells were treated with 10 ␮g/␮l acti- expression in MSCs and neuronal cells stimulated with nomycin, and then incubated with or without 10 nM SP. At 4-h intervals, up Tac1 to 36 h, total RNA was studied by Northern analysis for expression of IL-1RI inflammatory cytokines mRNA. Results are presented as band densities normalized with 18S rRNA. This section examines whether SP production by uninduced and induced MSCs stimulated with inflammatory cytokines (Figs. 3 and 4) correlates with expression of the Tac1 gene. Uninduced BAPTA-AM. All stimulated cells showed increased levels of SP MSCs and day 6 and 12 neuronal cells were stimulated with IL-1␣, with BAPTA treatment (Fig. 3B, p). The elevated levels are due to IL-2, IL-1␤, IL-6, or TNF-␣ and studied for Tac1 expression by the intracellular sequestration of SP by BAPTA through inhibition real-time RT-PCR. Uninduced MSCs stimulated with IL-1␣ or 3348 IL-1␣ IN MSC-DERIVED NEURONAL CELLS

Table I. Differential expression of genes linked to neurogenesis in IL-1␣-stimulated or unstimulated day 12 neuronal cellsa

D12 Cells

Gene Symbol Encoded Peptide No IL-1␣ IL-1␣ Fold Change Functions References

BDNF Brain-derived neurotrophic factor ϩϩϩϩ3.0 Neuronal survival 32 DVL3 Disheveled, dsh homolog 3 ϩϩϩϩ2.5 Neuroblast speciﬁcation 23 EGF Epidermal growth factor Ϫϩ ϩ16.0 Ectodermal mitogen 24 FEZ1 Fasciculation and elongation protein zeta 1 ϩϩϩϩ3.0 Axonal bundling and elongation 33 FGF2 Fibroblast growth factor 2 ϩϩϩϩ6.0 Ectodermal mitogen 25 LIMK1 LIM domain kinase 1 ϩϩϩϩ5.0 Brain development 26 MDK Midkine Ϫϩϩϩ16.0 Neurite growth factor 34 MT3 Metallothionein 3 ϩϩϩϩ2.0 Neuroprotection 35 NRG1 Neuregulin 1 Ϫϩ ϩ6.5 Neurotrophic factor 36 NRXN3 Neurexin 3 Ϫϩ ϩ11.0 CNS cell adhesion molecule 37 ROBO1 Roundabout 1 Ϫϩϩϩ13.5 Axonal guidance 27 SLIT2 Slit homolog 2 ϩϩϩϩ2.5 Axonal guidance 27 WNT1 Wingless-type, member 1 ϩϩϩϩ3.0 Brain development 28

a Total RNA from day 12 neuronal cells, stimulated with IL-1␣ or unstimulated, was used as a template to prepare biotin-UTP-labeled cRNA probes. The total RNA was pooled from three different donors. Probes were used for analyses of Oligo GEArray Human Neurogenesis and Neural Stem Cell Microarrays. The densities of spots were quantitated and then presented as fold change of stimulated/unstimulated. Details of the methods are presented in Materials and Methods. ϩ Fold change represents an increase Downloaded from in spot density in the stimulated cells.

IL-2 showed signiﬁcantly ( p Ͻ 0.05) increased Tac1 levels com- cess. Despite optimum concentration of 10 ng/ml IL-1␣ in SP pro- pared with unstimulated cells or cells stimulated with other cyto- duction, we asked whether lower concentrations can have effects kines (Fig. 5A). Signiﬁcantly ( p Ͻ 0.05) elevated levels of Tac1 on the neurogenic process. To this end, day 6, 9, and 12 neuronal were also observed in day 6 neuronal cells stimulated with TNF-␣ cells were stimulated for 16 h with IL-1␣ (1, 5 or 10 ng/ml), and

(Fig. 5B) and day 12 cells stimulated with IL-6 or TNF-␣ (Fig. then assayed for neuronal markers by Western analysis. Day 6 and http://www.jimmunol.org/ 5C). The results for IL-2, IL-6, and TNF-␣ correlate with the find- 9 cells were found to have decreased expression of the immature ings of the SP study (Fig. 4). Stimulation with IL-1␤ at all time neuronal marker, Nestin, with increasing concentrations of IL-1␣ points did not produce significantly ( p Ͼ 0.05) different Tac1 lev- (Fig. 7A, row 1). Day 12 cells no longer showed Nestin expression els compared with unstimulated cells (Fig. 5). Interestingly, day 6 at all concentrations of IL-1␣ (Fig. 7A, row 1). Day 9 and 12 cells and 12 cells stimulated with IL-1␣ did not produce significantly also showed increased expression of the mature neuronal marker, ( p Ͼ 0.05) different levels of Tac1 compared with unstimulated NeuN, with increasing IL-1␣ concentrations (Fig. 7A, row 2). cells (Fig. 5, B and C), even though both cells synthesize SP in IL-1␣ did not have any effect on NeuN expression in day 6 cells response to IL-1␣ (Fig. 3A). (Fig. 7A, row 2). Because IL-1␣ enhanced neuronal differentiation, we next de- by guest on September 26, 2021 Reuptake of SP in uninduced MSCs and neuronal cells termined whether IL-1␣ concomitantly decreased expression of Because IL-1␣-stimulated MSCs and neuronal cells release SP and stem cell-specific markers. Uninduced (day 0) MSCs were found express its receptor, NK1 (Figs. 3A and 4D), we examined whether to decrease expression of the stem cell-specific differentiation pro- the cells can reuptake SP from the growth medium. This question tein, Notch1, at increasing IL-1␣ concentrations of 5 and 10 ng/ml was addressed using fSP, because reuptake needs to be visually (Fig. 7B, row 1). Notch1 expression was not detected in day 6 or tracked. fSP was added to the cultures of uninduced MSCs and day 12 neuronal cells at any concentration of IL-1␣ (Fig. 7B, row 1). 12 neuronal cells at 0.1 and 1 ␮M. Neither concentration of fSP ␣ elicited reuptake in the uninduced cells (Fig. 6A, top panels). Day IL-1 enhances expression of neurogenesis-linked genes during 12 cells showed internalized fSP at 1 ␮M, but not 0.1 ␮M (Fig. 6A, neuronal induction middle panels). SP reuptake specificity was conferred by inhibition The final set of studies determined the global effect of IL-1␣ on in the presence of 10 ␮M unlabeled SP or CP-96,345, an NK1- MSC-derived neurogenesis. At induction, NIM was supplemented specific antagonist (Fig. 6A, bottom panels). The results implicate with 10 ng/ml IL-1␣. At day 12 induction, total RNA was ex- the potential formation of an autocrine loop through the recycling tracted and then analyzed with a Human Neurogenesis Microarray, of SP. which consisted of 263 gene-specific oligos that are involved in neurogenesis: differentiation, motility, cell cycle, and proliferation. Stabilization of IL-1RI mRNA by SP The analyses were done with total RNA, pooled from three human The next set of studies addressed whether SP can increase the donors. Genes that showed Ͼ1.5-fold change were considered sig- responsiveness of the neuronal cells to IL-1␣ by stabilizing IL-1RI nificant and are presented in Table I. The overall effect of supple- mRNA. Day 12 cells were treated with 10 ␮g/␮l actinomycin, to menting the NIM with IL-1␣ was the up-regulation of genes linked inhibit RNA transcription, and then incubated in the presence or to neuronal proliferation, development, and axonal guidance absence of 10 nM SP. At 4-h intervals, up to 36 h, cells were (23–28). studied for expression of IL-1RI mRNA by Northern analysis. Cells treated with SP stabilized IL-1RI mRNA (Fig. 6B, dotted Discussion line), while the untreated group degraded with time (Fig. 6B, solid One of the most challenging considerations for the clinical use of line). The results imply that SP can increase the responsiveness of stem cells is predicting how the recipient tissue will guide implan- day 12 neuronal cells to IL-1␣. tation and behavior. This study reports on an in vitro model to study how an inflammatory microenvironment, expected in ␣ IL-1 promotes neuronal differentiation of MSCs chronic and acute tissue insults, such as multiple sclerosis and The above studies focused on SP, so we next asked whether IL-1␣ spinal cord injury, affects undifferentiated and transdifferentiated has a more comprehensive effect on the transdifferentiation pro- MSCs. At this point, the ideal stage for implantation of MSCs is The Journal of Immunology 3349 unclear: undifferentiated vs partially differentiated vs fully differ- with many pro- and anti-inflammatory mediators. However, the entiated. Regardless, the cells will be placed in a milieu of inflam- primary use of these cytokines in this model was to demonstrate matory mediators. This issue is relevant even though particular the potential effects that an inflammatory milieu can have on stem diseases might have a special protocol for stem cell therapy. For cell implantation and behavior. The results from Figs. 3 and 4 this reason, our study examines the entire period of neuronal in- suggest that SP production is a valid readout to study the effects of duction to better predict the periods of differentiation that hold the inflammatory mediators during transdifferentiation into neuronal greatest clinical significance. cells. SP was chosen because it can be produced by both neuronal We began our timeline investigation by examining the expres- and non-neuronal cells and because it has immunomodulatory ef- sion of Tac1 during the course of neuronal induction. Our previous fects. For this reason, SP was ideal to examine how inflammatory report used stringent criteria as readout for transdifferentiation to mediators could potentially alter the behavior of transplantable neuronal cells, specifically microarray analyses, neuronal marker cells, whether undifferentiated, partially, or fully differentiated. expression, and electrophysiological activity (19). Tac1, and its The SP quantification studies demonstrate that the neuronal encoded peptide SP, was selected as a representative neurotrans- cells express Tac1 mRNA (Fig. 2), but do not produce SP unless mitter gene. Tac1 expression was shown to be significantly ( p Ͻ stimulated with IL-1␣ (Fig. 3). A plausible explanation for 0.05) up-regulated at days 6 and 12 induction, with no expression these results could be transient translational repression by mi- in the uninduced MSCs (Fig. 2). These studies were consistent croRNAs (miRNAs), because IL-1␣ stimulation of neuronal with the results from the reporter vector analyses (Fig. 1). Stable cells did not significantly increase Tac1 expression compared transfection of the vectors during the entire period of induction is with unstimulated cells (Fig. 5). miRNAs are a class of 19- to believed to result from the neuronal cells becoming postmitotic 23-nt small, noncoding RNAs, which function to cause mRNA Downloaded from (Fig. 1B). The vector construct, TAC1/NO, was examined for tran- degradation or transient translational repression depending on scriptional regulatory elements within exon 1 and intron 1 (Fig. target sequence complementarity (29). Treatment of the neuro- 1C). Interestingly, uninduced MSCs and day 6 neuronal cells ex- nal cells with cycloheximide, to inhibit protein synthesis, or hibited significantly ( p Ͻ 0.05) greater promoter activity in actinomycin, to inhibit RNA transcription would better deter- TAC1/NO as compared with TAC1–1.2 (Fig. 1C). The omitted mine the level of regulation. Preliminary data from our labora-

region of TAC1/NO has several regulatory elements that could be tory suggests a role for miRNAs in the observed results (our http://www.jimmunol.org/ potentially repressive to Tac1 transcription. Our ongoing studies unpublished observation). Uninduced MSCs do not express are focused on the RE-1 silencer of transcription binding site Tac1 (Fig. 2), but are capable of SP production (Fig. 3A). IL-1␣ within exon 1 as a main repressor of Tac1 expression. stimulation of uninduced cells resulted in elevated levels of We showed SP production in the transdifferentiated cells fol- Tac1 (Fig. 5A), thereby allowing production of SP. lowing stimulation with IL-1␣ (Fig. 3A). SP levels in the medium Day 12 neuronal cells were found to uptake SP from the growth were not due to stored internal SP, as indicated by treatment with medium at high SP concentration (Fig. 6A). SP induction of day 12 the calcium chelator, BAPTA-AM, which inhibits calcium-medi- cells was also shown to stabilize IL-1RI mRNA (Fig. 6B). These ated neurotransmitter release. Interestingly, the total levels of SP in results provide evidence that IL-1␣ stimulation could potentially the IL-1␣ stimulated, BAPTA-treated group (Fig. 3B, p) were form a positive feedback loop between SP and IL-1␣. Stimulation by guest on September 26, 2021 considerably less than the levels found in the culture medium of of day 12 cells causes SP production and internalization following the stimulated cells (Fig. 3A, s). An explanation for this discrep- receptor saturation (Fig. 6A). The high levels of SP can stabilize ancy may be incomplete calcium chelation, with release of SP into IL-1RI mRNA, and thus make the cells more responsive to IL-1␣ the growth medium, or enhanced activation of endogenous en- (Fig. 6B). To test this hypothesis, cells stimulated for longer pe- dopeptidases following accumulation of endogenous stores caused riods of time (Ͼ16 h) or stimulated multiple times should accu- by BAPTA. mulate more SP in the growth medium. The clinical implication of The stimulatory effects on SP production were not limited to a SP feedback loop could potentially be deleterious. In vivo, SP IL-1␣, because similar effects were observed in uninduced MSCs has a stimulatory effect on immune cell development and function for IL-2 and day 12 neuronal cells for IL-6 and TNF-␣ (Fig. 4). (30). Excessive production of SP by transplanted MSCs might lead These results were consistent with the levels of Tac1 mRNA to immune cell infiltration and transplant rejection. In places such present in each cell type (Fig. 5). Interestingly, these cytokines also as the brain or spinal cord, an exacerbated immune response could had a blunting effect on SP production in the other cell type. The be extremely damaging. lack of SP in neuronal cells stimulated with IL-2 cannot be ex- A surprising observation from these studies was the positive plained by SP reuptake (Fig. 6A), because stabilization of IL-1RI effect IL-1␣ had on neuronal differentiation. Stimulation of the mRNA by SP (Fig. 6B) should lead to more receptor availability neuronal cells for 16 h facilitated the up-regulation of mature neu- and further SP production. The blunting effects also cannot be ronal markers (NeuN; Fig. 7A). Inclusion of IL-1␣ within the NIM explained by down-regulation of the cytokines’ respective recep- enhanced the neurogenic program of differentiation and survival tors in uninduced or induced cells (Fig. 4D). Stable receptor ex- (Table I). These results have implications regarding the stage of pression during transdifferentiation indicates specific functions for differentiation in which to implant MSCs. If an inflammatory mi- the cytokines in both cell types. However, unlike IL-1␣, these croenvironment can be conducive to neuron-specific differentia- functions do not appear to include SP regulation at all time points. tion, the best time frame for implantation may be earlier than our Between days 0 and 12 of induction, we were comparing two experimental endpoint of day 12. significantly different cell types, thus suggesting that the mecha- One potential clinical dilemma that is an active avenue of re- nism of SP induction may also differ. Although an uninduced MSC search is the immune properties of the transplanted stem cells: Ag and a day 12 neuronal cell share a common cytokine receptor, it presentation and/or MHC class II (MHC-II) expression. MSCs ex- does not necessitate that the intracellular transduction mechanisms press MHC-II in response to low-level IFN-␥ and can function as involved in activating Tac1 transcription are similar. APCs (10, 31). 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