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The Journal of Immunology, 2007, 179, 3342 -3350
Copyright © 2007 by The American Association of Immunologists, Inc.
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Enhancing Effect of IL-1{alpha} on Neurogenesis from Adult Human Mesenchymal Stem Cells: Implication for Inflammatory Mediators in Regenerative Medicine1

Steven J. Greco* and Pranela Rameshwar2,{dagger}

* Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103; and {dagger} Department of Medicine, University of Medicine and Dentistry of New Jersey–New Jersey Medical School, Newark, NJ 07103


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 unless cells were stimulated with IL-1{alpha}. 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{alpha} 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{alpha} and other proinflammatory cytokines using production of the major Tac1 peptide, substance P (SP), as readout. Although IL-1{alpha} 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{alpha} also enhanced 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
There are ongoing research studies on the therapeutic use of stem cells, including clinical trials for various diseases (1, 2, 3). The successful application of stem cells in these and other trials will depend on the type of transplanted stem cell, patient health, disease modality, and the microenvironment of the recipient tissue. Both adult stem cells (ASCs)3 and embryonic stem cells are attractive candidates for regenerative therapies (4). However, their transition from "bench to bedside" has been slow, partly due to immune rejection and tumor formation (4). ASCs tend to be tissue-specific, thus indicating that their normal lineage development is limited to specialized cells within a particular tissue or organ of residence. It could be argued that this seeming specificity of ASCs limits their regenerative capacity as compared with embryonic stem cells. However, some ASCs are easily attainable and show reduced propensity to transform (5).

Mesenchymal stem cells (MSCs) are ASCs mostly found in the bone marrow (BM), surrounding the blood vessels and trabecula (6). Physiologically, MSCs exhibit a "gate-keeper" function in that they monitor afferent and efferent immune cell migration within the BM (7, 8). MSCs are attractive candidates in models of regenerative medicine given their ease in harvesting, isolation, and expansion (9). MSCs appear to bypass immune rejection through veto properties, thus making them candidates for allogeneic transplantation (10). MSCs show lineage-specific differentiation along osteogenic, chondrogenic, and adipogenic paths (11). MSCs have also been reported to transdifferentiate into cells of ectodermal and endodermal tissue (12, 13, 14, 15).

Disease modality and patient health are two key variables in the 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 clinical procedures, such as mathematical modeling, are at the forefront of personalized medicine (16). These models evaluate the patient’s personal profile and medical history, and mathematically relate it to the etiology of the disease or disorder to predict the appropriate treatment. A critical consideration in mathematical modeling of disease or injury is the surrounding microenvironment (17). In vitro, a MSC’s microenvironment can be closely monitored to favor stem cell growth and/or differentiation. In vivo, the transplanted MSCs are exposed to immune cells and mediators that could influence the cells’ behavior.

Human MSCs (hMSCs) can generate functional neuronal cells (18, 19). We have previously shown expression of a neurotransmitter gene, Tac1, following transdifferentiation, although translation did not occur unless the cells were stimulated with IL-1{alpha} (18). This report used this information as the basis to study how production of the Tac1-encoded neurotransmitter, substance P (SP), could serve as an indicator of the interaction between undifferentiated MSCs or MSC-derived neuronal cells and inflammatory mediators expected in the microenvironment of an injured tissue. IL-1{alpha} was selected to represent a prototypical inflammatory cytokine. This report shows the ability of IL-1{alpha} 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
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-1β, IL-6, TNF-{alpha}, and IL-2 were purchased from R&D Systems. 4',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. CP-96,345, an NK1-specific antagonist, was obtained from Pfizer. Recombinant human IL-1{alpha} 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, β-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-IL-1RI and rabbit anti-IL-2R were obtained from Santa Cruz Biotechnology. 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 vacuum-gas plasma treated, tissue-culture Falcon 3003 petri dishes (BD Biosciences). 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 ~80% confluent. After four cell passages, the adherent cells were symmetric, CD14–, CD29+, CD44+, CD34–, CD45–, SH2+, prolyl-4-hydroxylase– (10).

Neuronal induction of MSCs

At ~70–80% confluence, MSCs were trypsinized and then subcultured in 60-mm Falcon 3002 petri dishes or in Falcon 353046 6-well plates (BD Biosciences). For Western analyses, Northern analyses, RT-PCR, and microarray profiling, 104 MSCs were seeded in 60-mm tissue-culture dishes. For transfection studies, SP ELISA and SP reuptake microscopy, 103 MSCs were seeded in 6-well tissue-culture plates. All cells were allowed to adhere to the culture surface overnight in D10 medium. At 20% confluence, D10 medium was replaced with neuronal induction medium (NIM), which was comprised of Ham’s DMEM/F12, 2% FCS (Sigma-Aldrich), B27 supplement, 20 µM RA, and 12.5 ng/ml basic fibroblast 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 50% NIM after 4 days of induction yielded similar results. All experimental endpoints were performed with a maximal confluence of 70% to control for contact inhibition (19).

Vectors

The 5' flanking region of the Tac1 gene was cloned previously and analyzed (20). Fig. 1A shows the regions of various inserts as a cartoon. pGL3-TAC1–1.2: 740 bp upstream of exon 1, exon 1 and parts of intron 1; pGL3-TAC1/NO: 740 bp upstream of exon 1.


Figure 1
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  		FIGURE 1. Luciferase activity of Tac1 reporter genes in uninduced MSCs and neuronal cells (days 6 and 12 (D6 and D12)). A, Schematic 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 MSCs, and then grown in NIM for 12 days. At select time points, cells were examined for the vector ampicillin resistance gene by RT-PCR to confirm stable transfection. C, Luciferase activities of TAC1–1.2 and TAC1/NO in transfected, uninduced MSCs and D6 and D12 neuronal cells. Results are presented as the mean ± SD of normalized luciferase for five different experiments, each performed with a different BM donor. Normalizations were performed by comparing the ratio of luciferase per microgram of protein in cells transfected with vector alone, and setting this value to 1. *, p < 0.05 vs uninduced MSCs transfected with TAC1–1.2; **, p < 0.05 vs similar cells transfected with TAC1–1.2.

 
Transfection and reporter gene assay

Experimental vectors with luciferase-reporter gene inserts were transfected in 50% confluent MSCs using Effectene (Qiagen). At 48 h, cell-free lysates were prepared as described previously (20). For neuronal cells, at 48 h, Effectene was replaced with NIM, and cells were induced for 6 or 12 days. Luciferase activities in 10 µl of extracts were quantitated using the luciferase assay system (Promega). Normalizations were performed by comparing the ratio of luciferase per microgram of protein in cells transfected with vector alone, and setting this value to 1.

To detect stable expression of luciferase-reporter vectors in transfected cells, plasmid DNA was extracted using the Qiaprep Spin Miniprep kit (Qiagen). The extracted DNA served as DNA template for amplification of the pGL3-basic ampicillin resistance gene (Ampr) by PCR. The primers for Ampr span +3302/+3608 of the pGL3-basic vector (Promega). PCR was performed with the following primer pairs: (forward) 5'-ttt atc cgc ctc cat cca-3' and (reverse) 5'-tac gga tgg cat gac agt-3'. The cycling profile for Ampr (20 cycles) is: 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s with a final extension at 72°C for 10 min. PCR (10 µl) were separated by 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 µg) was reverse transcribed, and 200 ng of cDNA was used to amplify Tac1. For real-time PCR, Tac1 was amplified with the Platinum SYBR Green qPCR SuperMix-UDG Kit (Invitrogen Life Technologies). The primers for Tac1 span +60/+328 (NM_003182) with the following sequences: (forward) 5'-act gtc cgt cgc aaa atc-3' and (reverse) 5'-ggg cca ctt gtt ttt caa-3'. PCR were normalized by amplifying the same sample of cDNA with primers specific for GAPDH, (for semiquantitative PCR) or β-actin (for real-time PCR). The primers for GAPDH and β-actin span +212/+809 (NM_002046) and +842/+1037 (NM_001101), respectively, with the following sequences: GAPDH (forward) 5'-cca ccc atg gca aat tcc atg gca-3' and (reverse) 5'-tct aga cgg cag gtc agg tcc acc-3'; β-actin (forward) 5'-tgc cct gag gca ctc ttc-3' and (reverse) 5'-gtg cca ggg cag tga tct-3'. Real-time PCR were performed with a 7500 Real-Time PCR System (Applied Biosystems). The cycling profile for semiquantitative PCR for Tac1 (40 cycles) and GAPDH (30 cycles) was: 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s with a final extension at 72°C for 10 min. The cycling profile for real-time PCR for Tac1 (40 cycles) and β-actin (40 cycles) was: 94°C for 15 s and 60°C for 45 s. Gene expression analysis was performed using the 7500 System SDS Software (Applied Biosystems). Normalizations were performed by comparing the ratio of Tac1 expression in uninduced or induced cells to β-actin, and setting this value to 1. PCR were separated by electrophoresis as described above.

SP quantification

Uninduced and neuronally induced MSCs were stimulated for 16 h with 10 ng/ml IL-1{alpha}. This concentration of IL-1{alpha} was found to be optimal for SP quantification. In additional experiments, cells were stimulated for 16 h with the following: IL-2 (10 ng/ml), IL-1β (10 ng/ml), IL-6 (5 ng/ml), or TNF-{alpha} (100 ng/ml). Cell growth medium was collected and competitive ELISA was used to quantify SP production, as previously described (21). Briefly, 96-well plates were coated with a complex of streptavidin-biotinylated SP. Equal volumes (50 µl) of unknown samples and optimum rabbit anti-SP were added to quadruplicate wells. For intracellular SP quantification, 50 µl of cell-free, whole cell lysates treated with 1 µl of protease inhibitor mixture were used as the unknown. Bound anti-SP was detected with alkaline phosphatase-conjugated goat anti-rabbit IgG and 104 phosphatase substrate (Sigma-Aldrich). SP levels were calculated from a standard curve developed with OD at 405 nm vs 12 serial dilutions of known SP concentrations.

Western analysis

Whole cell or membrane extracts were prepared from neuronal cells or uninduced MSCs. Cells were trypsinized and washed in PBS (pH 7.4). After this, 30 µl of 1x lysis buffer (Promega) was added and the cells were subjected to freeze/thaw cycles in a dry ice/ethanol bath. Cell-free, whole cell lysates were obtained by centrifugation at 4000 x g for 5 min at 4°C. Total protein was determined with a Bio-Rad DC protein assay kit. Extracts (20 µg) were treated with 1 µl of protease inhibitor mixture, and analyzed by Western blots using 4–20% SDS-PAGE precast gels (Bio-Rad). Proteins were next transferred onto polyvinylidene difluoride membranes (PerkinElmer). Membranes were incubated overnight with primary Abs and then detected the following day by a 2-h incubation with HRP-conjugated IgG. All primary and secondary Abs were used at final dilutions of 1/1000 and 1/2000, respectively. HRP was developed with chemiluminescence detection reagent (PerkinElmer). The membranes were stripped with Restore Stripping Buffer (Pierce) for reprobing with other Abs.

SP labeling and reuptake

SP was labeled with FITC using the EZ-Label FITC Protein Labeling kit (Pierce Biotechnology), according to manufacturer’s recommended guidelines. Briefly, lyophilized SP was dissolved in borate buffer and then incubated for 1 h in dimethylformamide containing FITC fluorescent dye. Excess fluorescent dye was removed in PBS buffer using a Slide-A-Lyzer MINI Dialysis Unit. FITC-labeled SP (fSP) was then stored at –20°C at a stock concentration of 100 mM.

For reuptake studies, stock fSP was diluted in 0.1% PBS/BSA and added to uninduced MSCs and day 12 neuronal cells at final concentrations of 0.1 or 1 µM for 1 h at 37°C. To show SP specificity, day 12 cells were coincubated with 1 µM fSP and 10 µM unlabeled SP or 0.01 µM 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 were next counterlabeled with the nucleus-specific dye, DAPI, at a final concentration of 0.3 µM. Labeled cells were examined on a three-color fluorescent microscope (Nikon Instruments).

Northern analysis

Northern analysis for IL-1RI mRNA in neuronally induced MSCs was performed as described previously (22). Briefly, day 12 neuronal cells were treated with 10 µg/µl actinomycin and then incubated with or without 10 nM SP. At 4-h intervals, up to 36 h, total RNA was extracted and 10 µg of each sample was separated by electrophoresis in 1.2% agarose. RNA was transferred to nylon membranes (S & S Nytran) and then hybridized with IL-1RI-specific cDNA probe, randomly labeled with 3000 Ci/mmol [{alpha}-32P]dATP (DuPont/NEN). Probes were labeled with the Prime-IT II random primer kit (Stratagene). To normalize RNA loading, membranes were stripped and reprobed with cDNA for 18S rRNA. Hybrids were detected by exposures in a phosphoimager cassette (Molecular Dynamics), which was scanned at different times from 6 to 24 h with the Typhoon 9410 Molecular Imager phosphoimager system (Molecular Dynamics). The IL-1RI cDNA probe was prepared by RT-PCR using primers that span +1026/+1373, with the following sequences: (forward) 5'-aga ata cac atg gta tag-3' and (reverse) 5'-agt cat ccc ttc cat aaa-3'. The RNA template for RT-PCR was derived from MSCs induced for 16 h with 10 nM SP.

Biotinylated cRNA synthesis

Total RNA was extracted from day 12 neuronal cells using the RNAqueous-4PCR kit (Ambion). The procedure followed manufacturer’s specified guidelines. Biotinylated-UTP cRNA probes were synthesized from the extracted RNA using the TrueLabeling-AMP 2.0 kit from SuperArray. Briefly, cDNA was synthesized from 1 µg of total RNA at 50°C for 1 h. Next, the cDNA was incubated overnight at 37°C with RNA polymerase enzyme and 10 mM biotinylated-UTP (Roche) to synthesize labeled cRNA probes. cRNA was purified using the SuperArray Arraygrade cRNA Cleanup kit.

Microarray analysis

Oligo GEArray Human Neurogenesis and Neural Stem Cell Microarrays were purchased from SuperArray. The protocol for the microarray analyses followed manufacturer’s recommendations. Briefly, day 12 neuronal cell cRNA probes were hybridized to the microarrays overnight at 60°C with gentle rotation in a hybridization oven. The following day, alkaline phosphatase-conjugated streptavidin was bound to the arrays for chemiluminescent substrate detection by autoradiography. Array spot density and differential probe expression was calculated using SuperArray’s GEArray Expression Analysis Suite software. Spot density was normalized to select positive and negative controls spotted onto each array.

Statistical analysis

Statistical data analyses were performed with ANOVA and Tukey-Kramer multiple comparisons test. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Reporter gene activity of Tac1 fragments in uninduced MSCs and neuronal cells

The ideal stage of stem cell differentiation for tissue implantation, that is transplantation of undifferentiated or differentiated cells, is 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, with reporter gene vectors. Because these studies required long-term experiments, we first determined feasibility by transfecting uninduced MSCs with the pGL3 luciferase reporter containing the 5' flanking region of Tac1: TAC1–1.2 and TAC1/NO (Fig. 1A). Stability of pGL3 transfection was determined by PCR for Ampr using plasmid DNA extracted from cells subjected to neuronal induction. Bands were obtained up to day 12 at the predicted size of 300 bp (Fig. 1B). Reporter gene activity in uninduced MSCs and days 6 and 12 neuronal cells showed increased activity for both fragments as differentiation progressed (Fig. 1C). TAC1/N0, which lacks exon 1 and the 5' region of intron 1, yielded significantly (p < 0.05) greater luciferase activity in the uninduced and day 6 cells (Fig. 1C, Formula ) as compared with TAC1–1.2 (Fig. 1C, {square}). There was no significant (p > 0.05) difference between TAC1-NO and TAC1–1.2 at day 12 induction (Fig. 1C, right bars).

Endogenous expression of Tac1

This section determined whether the reporter gene activity (Fig. 1C) relates to endogenous expression of Tac1. To address this question, we performed semiquantitative RT-PCR for Tac1. Fig. 2A shows time-dependent increase in band intensities for Tac1 mRNA. Normalization with GAPDH showed an increase in band intensities at day 12 neuronal induction. To verify the results shown in Fig. 2A, we studied the mRNA using a quantitative method. Quantification by real-time RT-PCR showed significant (p < 0.05) increase in days 6 and 12 neuronal cells compared with uninduced MSCs (Fig. 2B). In summary, the results show increased levels of Tac1 mRNA during the course of neuronal induction.


Figure 2
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  		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 ± SD fold change for three different experiments, each performed with a different BM donor. Normalizations were performed by comparing the ratio of Tac1 expression in uninduced cells to β-actin, and setting this value to 1. *, p < 0.05 vs uninduced MSCs.

 
SP production in IL-1{alpha}-stimulated MSCs and neuronal cells

We assessed whether Tac1 expression correlated with production of the gene’s encoded peptide, SP. Uninduced MSCs and days 6 and 12 neuronal cells were stimulated with 10 ng/ml of the inflammatory cytokine, IL-1{alpha}, or were unstimulated. After 16 h, the culture medium was assayed for SP by ELISA. At all time points, IL-1{alpha} induced the production of significant (p < 0.05) SP as compared with the unstimulated cells (Fig. 3A).


Figure 3
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  		FIGURE 3. SP production in IL-1{alpha}-stimulated MSCs and neuronal cells. Uninduced MSCs and day 6 and 12 (D6 and D12) neuronal cells were stimulated with 10 ng/ml IL-1{alpha}, or unstimulated, in the presence or absence of 10 µM BAPTA-AM. Following stimulation, (A) medium or (B) whole cell lysate was collected from both cell subsets and SP levels were quantified by ELISA. Results are presented as the mean SP concentration ± SD for five different experiments, each performed with a different BM donor. *, p < 0.05 vs unstimulated cells.

 
Next, we evaluated whether SP levels in IL-1{alpha}-stimulated medium could be due to the release of intracellular SP stores or de novo production. To this end, whole cell lysates from uninduced MSCs and day 6 and 12 neuronal cells, stimulated with IL-1{alpha} or unstimulated, were studied. Low to undetectable SP was found in all unstimulated cells (Fig. 3B, {square}), thus signifying that the low levels in the medium (Fig. 3A, {square}) could not be due to internally stored SP. Low to undetectable SP was also found in all stimulated cells (Fig. 3B, Formula ), thus signifying production and release into the medium (Fig. 3A, Formula ).

To inhibit neurotransmitter release, cells were stimulated with IL-1{alpha} or unstimulated in the presence of the calcium chelator, BAPTA-AM. All stimulated cells showed increased levels of SP with BAPTA treatment (Fig. 3B, Formula ). The elevated levels are due to the intracellular sequestration of SP by BAPTA through inhibition of calcium-mediated transmitter release. The results verify that the effects of IL-1{alpha} are not due to the induction of SP release from internal stores.

SP production by IL-1β, IL-2, IL-6, and TNF-{alpha}

This section explores the specificity of IL-1{alpha} in SP production. To address this question, we first studied the response in SP production to other inflammatory cytokines, IL-2, IL-1β, IL-6, and TNF-{alpha}. Uninduced MSCs stimulated with IL-2 showed significantly (p < 0.05) increased SP levels compared with unstimulated cells or cells stimulated with other cytokines (Fig. 4A). SP levels were increased in TNF-{alpha}-stimulated day 6 neuronal cells (Fig. 4B) and day 12 cells stimulated with IL-6 or TNF-{alpha} (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-{alpha} in uninduced MSCs (Fig. 4A). SP production was not observed in uninduced or induced cells stimulated with IL-1β.


Figure 4
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  		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-1β, IL-6, TNF-{alpha}, or unstimulated, was collected and SP levels were quantified by ELISA. Results are presented as the mean SP concentration ± 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{alpha} or unstimulated, were prepared and analyzed by Western blot with anti-IL-1RI, -IL-6R, -TNFRI, -NK-1, and -IL-2R{alpha} Abs. Normalizations were performed with anti-β-actin Ab. Representative blot is shown for three different experiments, each performed with a different BM donor. *, p < 0.05 vs unstimulated cells.

 
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 were observed in uninduced and induced cells for IL-1RI, IL-6R, and TNF-RI (Fig. 4D, rows 2–4). We next determined whether the SP receptor, neurokinin 1 (NK1), was expressed, because this could signify possible reuptake of SP after IL-1{alpha} stimulation. We observed dense bands for NK1 at all time points (Fig. 4D, row 5). Stimulation of the cells with IL-1{alpha} had little to no effect on expression of the studied receptors (Fig. 4D).

Tac1 expression in MSCs and neuronal cells stimulated with inflammatory cytokines

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 MSCs and day 6 and 12 neuronal cells were stimulated with IL-1{alpha}, IL-2, IL-1β, IL-6, or TNF-{alpha} and studied for Tac1 expression by real-time RT-PCR. Uninduced MSCs stimulated with IL-1{alpha} or IL-2 showed significantly (p < 0.05) increased Tac1 levels compared with unstimulated cells or cells stimulated with other cytokines (Fig. 5A). Significantly (p < 0.05) elevated levels of Tac1 were also observed in day 6 neuronal cells stimulated with TNF-{alpha} (Fig. 5B) and day 12 cells stimulated with IL-6 or TNF-{alpha} (Fig. 5C). The results for IL-2, IL-6, and TNF-{alpha} correlate with the findings of the SP study (Fig. 4). Stimulation with IL-1β at all time points did not produce significantly (p > 0.05) different Tac1 levels compared with unstimulated cells (Fig. 5). Interestingly, day 6 and 12 cells stimulated with IL-1{alpha} did not produce significantly (p > 0.05) different levels of Tac1 compared with unstimulated cells (Fig. 5, B and C), even though both cells synthesize SP in response to IL-1{alpha} (Fig. 3A).


Figure 5
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  		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{alpha}, IL-2, IL-1β, IL-6, TNF-{alpha}, or unstimulated was studied for Tac1 expression by real-time RT-PCR. Results are presented as the mean ± SD fold change for three different experiments, each performed with a different BM donor. Normalizations were performed by comparing the ratio of Tac1 expression in unstimulated cells to β-actin, and setting this value to 1. *, p < 0.05 vs unstimulated cells.

 
Reuptake of SP in uninduced MSCs and neuronal cells

Because IL-1{alpha}-stimulated MSCs and neuronal cells release SP and express its receptor, NK1 (Figs. 3A and 4D), we examined whether the cells can reuptake SP from the growth medium. This question was addressed using fSP, because reuptake needs to be visually tracked. fSP was added to the cultures of uninduced MSCs and day 12 neuronal cells at 0.1 and 1 µM. Neither concentration of fSP elicited reuptake in the uninduced cells (Fig. 6A, top panels). Day 12 cells showed internalized fSP at 1 µM, but not 0.1 µM (Fig. 6A, middle panels). SP reuptake specificity was conferred by inhibition in the presence of 10 µM unlabeled SP or CP-96,345, an NK1-specific antagonist (Fig. 6A, bottom panels). The results implicate the potential formation of an autocrine loop through the recycling of SP.


Figure 6
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  		FIGURE 6. IL-1RI mRNA stabilization by internalized SP in day 12 (D12) neuronal cells. A, Uninduced MSCs and D12 neuronal cells were incubated with 0.1 µM (top and middle, left panels) or 1 µM fSP (top and middle, right panels), and then counterlabeled with nucleus-specific DAPI. SP-specific internalization in D12 cells was shown by coincubation with 10 µM unlabeled SP (bottom, left panel) or 0.01 µM SP receptor, NK1, antagonist (bottom, right panel). Figure shows representative labelings of three different experiments, each performed with a different BM donor. Images are shown at x20 magnification. B, D12 cells were treated with 10 µg/µl actinomycin, and then incubated with or without 10 nM SP. At 4-h intervals, up to 36 h, total RNA was studied by Northern analysis for expression of IL-1RI mRNA. Results are presented as band densities normalized with 18S rRNA.

 
Stabilization of IL-1RI mRNA by SP

The next set of studies addressed whether SP can increase the responsiveness of the neuronal cells to IL-1{alpha} by stabilizing IL-1RI mRNA. Day 12 cells were treated with 10 µg/µl actinomycin, to inhibit RNA transcription, and then incubated in the presence or absence of 10 nM SP. At 4-h intervals, up to 36 h, cells were studied for expression of IL-1RI mRNA by Northern analysis. Cells treated with SP stabilized IL-1RI mRNA (Fig. 6B, dotted line), while the untreated group degraded with time (Fig. 6B, solid line). The results imply that SP can increase the responsiveness of day 12 neuronal cells to IL-1{alpha}.

IL-1{alpha} promotes neuronal differentiation of MSCs

The above studies focused on SP, so we next asked whether IL-1{alpha} has a more comprehensive effect on the transdifferentiation process. Despite optimum concentration of 10 ng/ml IL-1{alpha} in SP production, we asked whether lower concentrations can have effects on the neurogenic process. To this end, day 6, 9, and 12 neuronal cells were stimulated for 16 h with IL-1{alpha} (1, 5 or 10 ng/ml), and then assayed for neuronal markers by Western analysis. Day 6 and 9 cells were found to have decreased expression of the immature neuronal marker, Nestin, with increasing concentrations of IL-1{alpha} (Fig. 7A, row 1). Day 12 cells no longer showed Nestin expression at all concentrations of IL-1{alpha} (Fig. 7A, row 1). Day 9 and 12 cells also showed increased expression of the mature neuronal marker, NeuN, with increasing IL-1{alpha} concentrations (Fig. 7A, row 2). IL-1{alpha} did not have any effect on NeuN expression in day 6 cells (Fig. 7A, row 2).


Figure 7
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  		FIGURE 7. IL-1{alpha} 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{alpha} (1, 5, and 10 ng/ml). Whole cell extracts were prepared and analyzed by Western blot with (A) anti-Nestin, -NeuN, and (B) -Notch1 Abs. Normalizations were performed with anti-β-actin Ab. Representative blots are shown for three different experiments, each performed with a different BM donor.

 
Because IL-1{alpha} enhanced neuronal differentiation, we next determined whether IL-1{alpha} concomitantly decreased expression of stem cell-specific markers. Uninduced (day 0) MSCs were found to decrease expression of the stem cell-specific differentiation protein, Notch1, at increasing IL-1{alpha} concentrations of 5 and 10 ng/ml (Fig. 7B, row 1). Notch1 expression was not detected in day 6 or 12 neuronal cells at any concentration of IL-1{alpha} (Fig. 7B, row 1).

IL-1{alpha} enhances expression of neurogenesis-linked genes during neuronal induction

The final set of studies determined the global effect of IL-1{alpha} on MSC-derived neurogenesis. At induction, NIM was supplemented with 10 ng/ml IL-1{alpha}. At day 12 induction, total RNA was extracted and then analyzed with a Human Neurogenesis Microarray, which consisted of 263 gene-specific oligos that are involved in neurogenesis: differentiation, motility, cell cycle, and proliferation. The analyses were done with total RNA, pooled from three human donors. Genes that showed >1.5-fold change were considered significant and are presented in Table I. The overall effect of supplementing the NIM with IL-1{alpha} was the up-regulation of genes linked to neuronal proliferation, development, and axonal guidance (23, 24, 25, 26, 27, 28).


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

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
One of the most challenging considerations for the clinical use of stem cells is predicting how the recipient tissue will guide implantation and behavior. This study reports on an in vitro model to study how an inflammatory microenvironment, expected in chronic and acute tissue insults, such as multiple sclerosis and spinal cord injury, affects undifferentiated and transdifferentiated MSCs. At this point, the ideal stage for implantation of MSCs is unclear: undifferentiated vs partially differentiated vs fully differentiated. Regardless, the cells will be placed in a milieu of inflammatory mediators. This issue is relevant even though particular diseases might have a special protocol for stem cell therapy. For this reason, our study examines the entire period of neuronal induction to better predict the periods of differentiation that hold the greatest clinical significance.

We began our timeline investigation by examining the expression of Tac1 during the course of neuronal induction. Our previous report used stringent criteria as readout for transdifferentiation to neuronal cells, specifically microarray analyses, neuronal marker expression, and electrophysiological activity (19). Tac1, and its encoded peptide SP, was selected as a representative neurotransmitter gene. Tac1 expression was shown to be significantly (p < 0.05) up-regulated at days 6 and 12 induction, with no expression in the uninduced MSCs (Fig. 2). These studies were consistent with the results from the reporter vector analyses (Fig. 1). Stable transfection of the vectors during the entire period of induction is believed to result from the neuronal cells becoming postmitotic (Fig. 1B). The vector construct, TAC1/NO, was examined for transcriptional regulatory elements within exon 1 and intron 1 (Fig. 1C). Interestingly, uninduced MSCs and day 6 neuronal cells exhibited significantly (p < 0.05) greater promoter activity in TAC1/NO as compared with TAC1–1.2 (Fig. 1C). The omitted region of TAC1/NO has several regulatory elements that could be potentially repressive to Tac1 transcription. Our ongoing studies are focused on the RE-1 silencer of transcription binding site within exon 1 as a main repressor of Tac1 expression.

We showed SP production in the transdifferentiated cells following stimulation with IL-1{alpha} (Fig. 3A). SP levels in the medium were not due to stored internal SP, as indicated by treatment with the calcium chelator, BAPTA-AM, which inhibits calcium-mediated neurotransmitter release. Interestingly, the total levels of SP in the IL-1{alpha} stimulated, BAPTA-treated group (Fig. 3B, Formula ) were considerably less than the levels found in the culture medium of the stimulated cells (Fig. 3A, Formula ). An explanation for this discrepancy may be incomplete calcium chelation, with release of SP into the growth medium, or enhanced activation of endogenous endopeptidases following accumulation of endogenous stores caused by BAPTA.

The stimulatory effects on SP production were not limited to IL-1{alpha}, because similar effects were observed in uninduced MSCs for IL-2 and day 12 neuronal cells for IL-6 and TNF-{alpha} (Fig. 4). These results were consistent with the levels of Tac1 mRNA present in each cell type (Fig. 5). Interestingly, these cytokines also had a blunting effect on SP production in the other cell type. The lack of SP in neuronal cells stimulated with IL-2 cannot be explained by SP reuptake (Fig. 6A), because stabilization of IL-1RI mRNA by SP (Fig. 6B) should lead to more receptor availability and further SP production. The blunting effects also cannot be explained by down-regulation of the cytokines’ respective receptors in uninduced or induced cells (Fig. 4D). Stable receptor expression during transdifferentiation indicates specific functions for the cytokines in both cell types. However, unlike IL-1{alpha}, these functions do not appear to include SP regulation at all time points. Between days 0 and 12 of induction, we were comparing two significantly different cell types, thus suggesting that the mechanism of SP induction may also differ. Although an uninduced MSC and a day 12 neuronal cell share a common cytokine receptor, it does not necessitate that the intracellular transduction mechanisms involved in activating Tac1 transcription are similar.

The individual use of the studied cytokines as a representation of an inflammatory microenvironment could be viewed as an oversimplification, because a diseased or injured tissue may be laden with many pro- and anti-inflammatory mediators. However, the primary use of these cytokines in this model was to demonstrate the potential effects that an inflammatory milieu can have on stem cell implantation and behavior. The results from Figs. 3 and 4 suggest that SP production is a valid readout to study the effects of inflammatory mediators during transdifferentiation into neuronal cells. SP was chosen because it can be produced by both neuronal and non-neuronal cells and because it has immunomodulatory effects. For this reason, SP was ideal to examine how inflammatory mediators could potentially alter the behavior of transplantable cells, whether undifferentiated, partially, or fully differentiated.

The SP quantification studies demonstrate that the neuronal cells express Tac1 mRNA (Fig. 2), but do not produce SP unless stimulated with IL-1{alpha} (Fig. 3). A plausible explanation for these results could be transient translational repression by microRNAs (miRNAs), because IL-1{alpha} stimulation of neuronal cells did not significantly increase Tac1 expression compared with unstimulated cells (Fig. 5). miRNAs are a class of 19- to 23-nt small, noncoding RNAs, which function to cause mRNA degradation or transient translational repression depending on target sequence complementarity (29). Treatment of the neuronal cells with cycloheximide, to inhibit protein synthesis, or actinomycin, to inhibit RNA transcription would better determine the level of regulation. Preliminary data from our laboratory suggests a role for miRNAs in the observed results (our unpublished observation). Uninduced MSCs do not express Tac1 (Fig. 2), but are capable of SP production (Fig. 3A). IL-1{alpha} stimulation of uninduced cells resulted in elevated levels of Tac1 (Fig. 5A), thereby allowing production of SP.

Day 12 neuronal cells were found to uptake SP from the growth medium at high SP concentration (Fig. 6A). SP induction of day 12 cells was also shown to stabilize IL-1RI mRNA (Fig. 6B). These results provide evidence that IL-1{alpha} stimulation could potentially form a positive feedback loop between SP and IL-1{alpha}. Stimulation of day 12 cells causes SP production and internalization following receptor saturation (Fig. 6A). The high levels of SP can stabilize IL-1RI mRNA, and thus make the cells more responsive to IL-1{alpha} (Fig. 6B). To test this hypothesis, cells stimulated for longer periods of time (>16 h) or stimulated multiple times should accumulate more SP in the growth medium. The clinical implication of a SP feedback loop could potentially be deleterious. In vivo, SP has a stimulatory effect on immune cell development and function (30). Excessive production of SP by transplanted MSCs might lead to immune cell infiltration and transplant rejection. In places such as the brain or spinal cord, an exacerbated immune response could be extremely damaging.

A surprising observation from these studies was the positive effect IL-1{alpha} had on neuronal differentiation. Stimulation of the neuronal cells for 16 h facilitated the up-regulation of mature neuronal markers (NeuN; Fig. 7A). Inclusion of IL-1{alpha} within the NIM enhanced the neurogenic program of differentiation and survival (Table I). These results have implications regarding the stage of differentiation in which to implant MSCs. If an inflammatory microenvironment can be conducive to neuron-specific differentiation, the best time frame for implantation may be earlier than our experimental endpoint of day 12.

One potential clinical dilemma that is an active avenue of research is the immune properties of the transplanted stem cells: Ag presentation and/or MHC class II (MHC-II) expression. MSCs express MHC-II in response to low-level IFN-{gamma} and can function as APCs (10, 31). We are currently investigating whether the day 12 neuronal cells can re-express MHC-II upon exposure to proinflammatory cytokines. If they do, the implanted cells could be rejected unless the host can tolerate the donor MHC-II. Given the diverse etiologies of insults characterized by an inflammatory environment, the methods of stem cell therapy may vastly differ.

The difficulty in predicting how a transplanted stem cell will behave in vivo underscores the necessity in the field to develop in vitro models that mimic the microenvironment of an injured tissue. To properly examine the effect of the microenvironment on the transplantable cell requires extensive experiments under a variety of conditions. Moreover, the problem becomes more complex when one considers the dynamic nature of the microenvironment and the differentiation potential of the implanted cell. The dynamic nature of the microenvironment is mediated by local cells and infiltrating immune cells which synthesize cytokines, chemokines, and matrix metalloproteases. These considerations pose a formidable opponent to the success of regenerative medicine, because vastly different outcomes could occur depending on the stage of differentiation of the transplanted stem cell.

Our studies report on an in vitro model to better understand how an inflammatory microenvironment regulates MSC-derived neurogenesis. Future studies are necessary to better model the inflammatory microenvironment through the inclusion of other pro- and anti-inflammatory mediators or coincubation with immune cells. Through like approaches, the risks associated with stem cell therapy can be better evaluated to prevent unforeseen harm to the patient.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the F. M. Kirby Foundation. This work 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 and Dentistry of New Jersey-New Jersey Medical School (Newark, NJ). Back

2 Address correspondence and reprint requests to Dr. Pranela Rameshwar, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Medical Sciences Building, Room E-579, 185 South Orange Avenue, Newark, NJ 07103. E-mail address: rameshwa{at}umdnj.edu Back

3 Abbreviations used in this paper: ASC, adult stem cell; MSC, mesenchymal stem cell; BM, bone marrow; SP, substance P; hMSC, human MSC; DAPI, 4',6-diamidino-2-phenylindole, dilactate; NIM, neuronal induction medium; fSP, FITC-conjugated SP; miRNA, microRNA; MHC-II, MHC class II; RA, retinoic acid. Back

Received for publication February 28, 2007. Accepted for publication June 27, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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