Immunostimulatory Effects of Mesenchymal Stem Cell‐Derived Neurons: Implications for Stem Cell Therapy in Allogeneic Transplantations

Abstract

Mesenchymal stem cells (MSCs) differentiate along various lineages to specialized mesodermal cells and also transdifferentiate into cells such as ectodermal neurons. MSCs are among the leading adult stem cells for application in regenerative medicine. Advantages include their immune‐suppressive properties and reduced ethical concerns. MSCs also show immune‐enhancing functions. Major histocompatibility complex II (MHC‐II) is expected to be downregulated in MSCs during neurogenesis. Ideally, “off the shelf” MSCs would be suited for rapid delivery into patients. The question is whether these MSC‐derived neurons can reexpress MHC‐II in a milieu of inflammation. Western analyses demonstrated gradual decrease in MHC‐II during neurogenesis, which correlated with the expression of nuclear CIITA, the master regulator of MHC‐II expression. MHC‐II expression was reversed by exogenous IFNY. One‐way mixed lymphocyte reaction with partly differentiated neurons showed a stimulatory effect, which was partly explained by the release of the proinflammatory neurotransmitter substance P (SP), cytokines, and decreases in miR‐130a and miR‐206. The anti‐inflammatory neurotransmitters VIP and CGRP were decreased at the peak time of immune stimulation. In summary, MSC‐derived neurons show decreased MHC‐II expression, which could be reexpressed by IFNY. The release of neurotransmitters could be involved in initiating inflammation, underscoring the relevance of immune responses as consideration for stem cell therapies.

Introduction

The postnatal bone marrow is host to two major stem cells: hematopoietic (HSCs) and mesenchymal (MSCs). ¹ , ² , ³ Although MSCs are present in other organs, the bone marrow remains the major site of residence. ⁴ , ⁵ The anatomical location of MSCs is distinct from HSCs. The latter is located close to the endosteum, whereas MSCs are found around the vasculature system, and in contact with the trabeculae. ¹ , ² , ⁶ HSCs and MSCs are both mesodermal cells that differentiate along multiple lineages to generate specialized cells belonging to the same germ layer. ⁷ , ⁸ The cells formed by HSCs and MSCs are distinct. The former generates blood and immune cells and the latter generates cells such as adipocytes, chondrocytes, and stromal cells. ² , ³ , ⁹ Since MSCs have been shown to transdifferentiate into ectodermal and endodermal cells, these stem cells are considered to be plastic. ¹⁰ , ¹¹ , ¹²

The plastic nature of MSCs, combined with their ease of expansion from adult bone marrow aspirates and other sources, support their candidacy for stem cell therapies. MSCs express major histocompatibility complex II (MHC‐II), which raises concerns of rejection in settings of allogeneic transplantations. This fear has been partly alleviated by the ability of MSCs to exert immune‐suppressive properties. ³ , ¹³ , ¹⁴ , ¹⁵ The mechanisms by which MSCs mediate immune suppression are complex and could be partly explained by their veto function, blunting of immune cell development, negative feedback on antigen‐presenting cells (APCs), and the production of antiinflammatory cytokines. ¹³ , ¹⁶ , ¹⁷ In contrast to their immune‐suppressive properties, MSCs exhibit proinflammatory functions. They have been shown to act as phagocytes and APCs, and are involved in T‐cytotoxic (CTL) responses to viral infection, although at reduced efficiency. ¹³ , ¹⁵ , ¹⁸ IFNγ exerts bimodal effects on MHC‐II expression in MSCs, with increased expression at low IFNγ levels and decreased expression at high levels. ¹³ , ¹⁶ , ¹⁷

MSCs have been shown to generate functional dopamine cells, and other types of neurons. ¹² , ¹⁹ , ²⁰ , ²¹ The neurons express receptors for various cytokines and have been shown to respond to proinflammatory mediators. ²¹ , ²² The most efficient method to use MSCs in therapies for neural injuries is the delivery of available stem cells, which would be likely from allogeneic donors. The question is whether MHC‐II expression is retained on MSCs following transdifferentiation to neurons. On the other hand, if MHC‐II is decreased in the MSC‐derived neurons, could MHC‐II be reexpressed? This study reports on the induction of allogeneic peripheral blood mononuclear cell proliferation. The study further implicates neurotransmitter release as part of the reason for cell proliferation. Mature neurons are shown to be capable of reexpressing MHC‐II in the presence of IFNγ. The implication for allogeneic transplantation of MSCs or the transdifferentiated neurons is discussed.

Materials and Methods

Human subjects

The use of these human samples was approved by the Institutional Review Board of the University of Medicine and Dentistry of New Jersey (UMDNJ), Newark Campus. Peripheral blood (PB) and BM aspirates were obtained from healthy subjects between ages 18 and 30 years. BM aspirates were taken from the posterior iliac crest and immediately preserved in heparin‐free media. Mononuclear cells were isolated from PB (PBMCs) by Ficoll Hypaque Density gradient.

Reagents

NK1 antagonist CP‐99,994 was kindly provided by Pfizer (Groton, CT, USA). The antagonist was stored at −80°C in dimethyl sulph‐oxide at 0.1 M in 10‐μL aliquots and then stored at −80°C for up to 1 month. Immediately before an assay, the NK1 antagonist was diluted in serum‐free media.

DMEM with high glucose, DMEM/F12, L‐glutamine, and B‐27 supplement were purchased from Gibco (Carlsbad, CA, USA). Fetal calf sera (FCS) were purchased from Sigma (St Louis, MO, USA). Defined FCS was purchased from Atlanta Biologicals (Lawrenceville, GA, USA). All‐trans retinoic acid (RA), endotoxin low PBS (pH 7.4), and Ficoll‐Hypaque were purchased from Sigma.

4′, 6‐diamidino‐2‐phenylindole, dilactate (DAPI) was purchased from Molecular Probes (Carlsbad, CA, USA). 1,2‐bis‐(0‐Aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetra‐(acetoxymethyl) ester (BAPTA‐AM) was purchased from Calbiochem (http://www.calbiochem.com). BAPTA‐AM was dissolved in sterile DMSO at 10 mM and then purged with nitrogen. After this, the solution was aliquoted in 20‐μL volumes and then stored at −20°C. Texas Red Phalloidin (F‐actin) was obtained from Molecular Probes.

Antibodies and cytokines

Recombinant human basic fibroblast growth factor (bFGF) was purchased from R&D Systems (Minneapolis, MN, USA), β‐actin mAb from Sigma, FITC‐conjugated goat anti‐mouse from Jackson ImmunoResearch (West Grove, PA, USA), and FITC mouse anti‐goat IgG and PE‐conjugated IgG isotype controls from Becton Dickinson (B&D) (San Jose, CA, USA). The following antibody was purchased from BD PharMingen (San Jose, CA, USA): PE‐conjugated HLA‐DR mAb. IFNγ was obtained from Collaborative Research Incorporated (Bedford, MA, USA). Goat anti‐CIITA (N‐20) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and rabbit anti‐CD45 was obtained from Abcam (Cambridge, MA, USA). Rabbit anti‐calcitonin gene‐related peptide (CGRP) was purchased from Chemicon International (Temecula, CA, USA). Monoclonal vasoactive intestinal peptide (VIP) antibody was obtained from Cure Gastroenteric Biology Center (Los Angeles, CA, USA). FITC‐anti‐goat IgG and PE‐anti‐rabbit IgG were purchased from Open Biosystems (Huntsville, AL, USA), FITC‐anti‐mouse IgG from Abcam, and rabbit anti‐acetyl‐histone H3 from Upstate (Lake Placid, NY, USA).

The MHC‐II hybridoma (IVA12) was purchased from American Type Culture Collection (Manassas, VA, USA) and grown according to their instructions. IVA12 cells were injected i.p. in BALB/c mice that were >6 months old. Mice were housed in an American Association for Accreditation of Laboratory Animal Care‐accredited animal facility, and the use of mice for propagation of ascites was approved by the UMDNJ institutional animal care and use committee. MHC‐II Abs were collected as ascites and then purified by affinity on a Sepharose G column. Purified IgG was tested against MSC in dose‐binding immunofluorescence assays. Nonspecific binding was determined with fibroblasts, differentiated from MSCs.

Culture of MSC

MSCs were cultured from BM aspirates as described. ¹⁵ Briefly, unfractionated aspirates (2 mL) were added to D10 media, which consisted of DMEM and 10% defined FCS (Premium). The cell suspension was transferred to plasma‐treated, tissue culture Falcon 3003 petri dishes (Fisher Scientific, Springfield, NJ, USA). Plates were incubated for 3 days after which red blood cells and granulocytes were removed by Ficoll Hypaque density gradient. Fifty percent of media were replaced with fresh D10 media at weekly intervals until the adherent cells were approximately 80% confluent. After four cell passages, the adherent cells were symmetric, CD14⁻, CD29⁺, CD44⁺, CD34⁻, CD45⁻, SH2⁺ (CD105), prolyl‐4‐hydroxylase⁻. ¹⁵

Neuronal induction of MSCs

MSCs were induced to neuronal cells as described. ²² Briefly, 10³ MSCs were seeded in D10 media. After adherence at 20% confluence, the D10 media was replaced with neuronal induction media, which comprises Ham's DMEM/F12, 2% FCS (Sigma), B27 supplement, 20 μM RA, and 12.5 ng/mL basic FGF. The media were unchanged during the induction periods.

One‐way mixed lymphocyte reaction (MLR)

Stimulator cells were prepared by inducing MSCs at different times with neuronal induction media (described above). Induction was done with MSCs (3×10³) for 4, 6, 8, and 12 days, referred as D4, D6, D8, D12, respectively. Uninduced MSCs served as controls. After the induction time, media were aspirated and the wells were washed twice with PBS (pH 7.4).

Responder PBMCs were suspended at 2×10⁶ cells/mL in D10 media. At 72 hours of culture, each well was pulsed with 1 μCi/well of [methyl‐³H] thymidine (TdR) (70–90 Ci/mmol (NEN, Boston, MA). After 16 hours, the cells were harvested onto glass‐fiber filters (Cambridge Technologies, Cambridge, MA, USA). [³H]TdR incorporation was quantified in a scintillation counter (Beckman, Fullerton, CA, USA). The results are expressed as the mean DPM of experimental cultures (responders + stimulators).

Flow cytometry

Uninduced MSCs and 6‐ and 12‐day‐induced MSCs were seeded in 600‐mL Falcon flasks (35–5001). At 80% confluence, MSCs were de‐adhered with dissociation solution, washed with 1× PBS (pH 7.4), and then incubated for 30 minutes at room temperature with PE anti–HLA‐DR (MHC‐II) at a final concentration of 1:500. Isotype controls were incubated with mouse IgG conjugated to PE. Cells were fixed with 0.4% paraformaldehyde and then analyzed by FACScan (FACS Caliber; Becton Dickinson, Franklin Lakes, NJ, USA).

Immunofluorescence microscopy

MSCs at 2 × 10³, uninduced and induced for different times, were seeded on cover slips placed in 12‐well plates (Falcon 3043). The adherent cells were washed twice with PBB, which consists of 1X PBS containing 0.1% bovine serum albumin. The cells were fixed with 3.7% paraformaldehyde for 20 minutes and then washed with PBB. Cells were permeabilized with PBB containing 0.1% Triton X for 45 minutes. After this, cells were washed twice with PBB and then incubated with primary antibodies overnight at 4°C, at the following dilutions: CIITA, 1/500; MHC‐II, 1/500, and CD45, 1/500. This was followed by incubation at room temperature for 1 hour with the respective secondary antibodies at 1/1,000 final dilution. Cells were washed twice with PBB and cytoskeletal staining was developed with Texas Red Phalloidin (F‐actin) at 6.0 μM final dilution. Nuclear visualization was developed with DAPI at a dilution of 300 nM.

Western blot

Induced (4, 8, 12) and uninduced MSCs were stimulated with 10 U/mL of IFNγ. After 16 hours, whole cell extracts were prepared as described. ²³ Briefly, cells were washed with PBS and then lysed with 1× Lysis Buffer (Promega, Madison, WI, USA). The cells were dissociated with trypsin and then subjected to freeze/thaw cycles in a dry ice/ethanol bath. Cell‐free whole cell lysates were obtained by centrifugation at 4,000 g for 5 minutes at 4°C. Total protein was determined with a Bio‐Rad DC protein assay kit (Bio‐Rad, Hercules, CA, USA). Nuclear and cytoplasmic extracts were prepared with the N‐Extracts (Sigma). Extracts (200 μg) were analyzed by Western blots using 4–20% SDS‐PAGE precast gels (Bio‐Rad). The proteins were transferred onto polyvinylidene difluoride membranes (Perkin Elmer Life Sciences, Boston, MA, USA). Membranes were incubated overnight at 4°C with primary antibodies and then detected the following day by 2‐hour incubation with HRP‐conjugated IgG. All primary and secondary antibodies were used at final dilutions of 1/500 and 1/2,000, respectively. HRP was developed with chemiluminescence detection reagent (Perkin Elmer Life Sciences). The membranes were stripped with Restore Stripping Buffer (Pierce, Rockford, IL, USA) for reprobing with other antibodies.

Substance P (SP) ELISA

SP was quantitated with a competitive ELISA as described. ²² Briefly, 96‐well plates were coated with a complex of streptavidin‐biotinylated SP. Equal volumes (50 μL) of the samples and optimum rabbit anti‐SP were added to triplicate wells. Bound anti‐SP was detected with AP‐conjugated goat anti‐rabbit IgG and Sigma 104 phosphatase substrate. SP levels were calculated from a standard curve developed with OD at 405 nm versus 12 serial dilutions of known SP concentrations.

Microarray for cytokines

Cell‐free media from MLR reactions with uninduced and induced (8‐ and 12‐day) MSCs were studied for cytokine expressions with human cytokine protein array III (Ray Biotech, Norcross, GA, USA), as described. ²⁴ In parallel analyses, media were studied from PBMCs cultured alone. Briefly, membranes were incubated for 1 hour with media and then incubated consecutively with biotin‐conjugated anticytokines and HRP‐streptavidin followed by chemiluminescence detection. Background was subtracted in analyses with fresh culture media.

Band density analysis

Protein densities were analyzed with the UN‐SCAN‐IT gel software (Silk Scientific, Orem, UT, USA). Bands were normalized with the bands for the respective housing‐keeping genes.

Real‐time RT‐PCR

The nonadherent cells were removed and the CD45‐negative cells were selected by magnetic bead isolated. The negatively selected cells were subjected to real‐time RT‐PCR for miR‐130a and miR‐206 with 2 μg of total RNA as described. ²⁵ The RNA was subjected to reverse transcription and then amplified by the mirVana qRT‐PCR miRNA Detection Kit (Ambion, Austin, TX, USA), according to the manufacturer's specified guidelines. Primers for miR‐130a (Accession No. MI00004) and miR‐206 (Accession No. MI0000490) were purchased from Ambion. PCRs were normalized with the same test samples using 5S rRNA primers (Accession No. V00589, Ambion). The normalization values were arbitrarily assigned 1. Amplifications were done with the Platinum SYBR Green qPCR SuperMix‐UDG Kit (Invitrogen) in a 7,500 Real‐time PCR System (Applied Biosystems, Foster City, CA). Gene expression analysis was performed using the 7,500 System SDS Software (Applied Biosystems).

Statistical analysis

Data were analyzed using analysis of variance and Tukey‐Kramer multiple comparisons test. p values of < 0.05 were considered significant.

Results

MHC‐II expression on MSC‐derived neurons

We have reported robust methods to generate functional neurons from human MSCs. ¹² , ¹⁹ The morphological differences between uninduced and induced (6‐ and 12‐day) MSCs are represented in Figure 1A. While the uninduced MSCs are morphologically long and spindle‐shaped symmetrical cells, the induced cells are morphologically distinct, with asymmetry and extensions of long thin processes from the cell bodies. Day 12‐induced cells are functional based on electrophysiology studies. ¹² , ¹⁹ Despite the formation of neurons, it is unclear if MHC‐II expression is developmentally changed as the MSCs form neurons. To this end, four different experiments were done to analyze MHC‐II expression on MSCs, uninduced and induced for 6 and 12 days. The cells were disassociated and then studied for MHC‐II expression by flow cytometry and the results are presented as mean fluorescence intensities (MFI ± SD). We observed significant (p < 0.05) decrease by day 6 and 12 inductions, as compared to uninduced and isotype control (Figure 1B).

Figure 1.

Expression of MHC on transdifferentiating MSCs to neurons. (A) Representative MSCs, uninduced or induced for 6 (D6) and 12 (D12) days. (B) Flow cytometry for MHC‐II (PE‐HLA‐DR) with MSCs (uninduced, Day 0) or induced for 6 and 12 days. Top Panel: Isotype control. Inset: Mean Immunofluorescence (MFI) ± SD, n = 4. (C) Representative of three Western blots for MHC‐I performed with cell membrane extracts from MSCs (uninduced, 0) or induced for various times. Membranes were stripped and reprobed with anti‐β‐actin for normalization. In all figures, each experiment was done with MSCs from a different donor. (D) Mean ± SD of the normalized band densities.

MHC‐I expression is not expected to change in the generated neurons. ²⁶ Since the field of transdifferentiation is new and has implications for translational science, it was unclear if MHC‐I expression would be changed. To this end, its expression was studied by Western blots with membrane extracts from MSCs, uninduced and induced for different times. Normalizations were studied by stripping and reprobing with anti‐β‐actin. The results indicate no significant change in MHC‐I expression between induced and uninduced MSCs (Figure 1C and 1D). In summary, the results showed decreased expression of MHC‐II following transdifferention to neurons, but no change in MHC‐I.

CIITA expression in uninduced and induced MSCs

CIITA transcription factor is a master regulator of MHC‐II expression. ²⁷ We therefore asked whether the changes in MHC‐II expression during neuronal induction could be explained by the lack of nuclear CIITA. This question was studied in time‐course immunofluorescence analyses with FITC‐anti‐CIITA and F‐actin for cytoskeleton visualization. The studies showed CIITA localization in the nuclei in uninduced MSCs (time 0) up to 6‐day induction (Figure 2A, white arrows). Cytoplasmic CIITA was detectable in 4‐day‐induced cells (Figure 2A, white arrows). At 8‐ and 12‐day induction, CIITA was only detected in the cytosol (Figure 2A). Nonimmune IgG and secondary antibodies alone showed no labeling. The results show correlations between CIITA and MHC‐II expression in MSCs subjected to neuronal induction. Membrane MHC‐II correlated with the presence of nuclear CIITA, whereas undetectable membrane MHC‐II correlated with the presence of cytosolic CIITA.

Figure 2.

Intracellular expression of CIITA in MSCs, uninduced MSCs (0) or induced for different times. (A) MSCs were induced on glass coverslips and then subjected to intracellular immunofluorescence with FITC‐anti‐CIITA. The cells were counterstained with F‐actin (red). Arrows indicate labelings for CIITA Figure represents three different experiments, each performed with a different donor. (B and C) The experiments described in A were repeated and the nuclear (B) and cytoplasmic (C) extracts from the MSCs (induced: 8 and 12 days; uninduced: 0) were analyzed by Western blots for CIITA. The membranes were stripped and reprobed with anti‐β‐actin (cytopsolic extracts) and Histone H3 (nuclear extracts). The studies show representative of three experiments. The normalized densities of the bands (mean ± SD), are presented in D and E.

We next verified the observations in Figure 2A by Western analyses. The experiments were repeated and the uninduced and induced MSCs were analyzed by Western blots using nuclear and cytoplasmic extracts. The result showed decreases in band intensities in the nuclear extracts for 6‐ and 12‐day‐induced MSCs (Figure 2B and 2E). This decrease correlated with increased band densities in the cytoplasmic extracts (Figure 2C and 2E). In both cases, the membranes were stripped and reprobed with anti‐β‐actin or with anti‐Histone H3. The reason for the latter is to verify that the cytoplasmic extracts were not contaminated with nuclear proteins. The blots are presented with normalized proteins: β‐actin for cytoplasmic and Histone H3 for nuclear extracts.

Proliferation of PBMCs in the presence of allogeneic uninduced and induced MSCs

Presently, scientific evidence has not proven if MSCs should be administered as stem cells, or as partly or fully transdifferentiated cells. While reduced expression of MHC‐II is a significant indicator for reduced graft‐versus‐host response, the question is whether the decrease, even if significant by day 6 (Figure 1B), is sufficient to prevent graft‐versus‐host response, which could be indicated by the MSCs and the derived neurons mediating PBMC proliferation. Uninduced MSCs have been previously shown to elicit allogeneic responses in one‐way MLR. ¹⁵ The proliferation of allogeneic PBMCs to MSC‐derived neurons was determined in one‐way MLR.

As expected, stimulator uninduced MSCs mediated the proliferation of PBMCs from unrelated donors (Figure 3, open bar). ¹⁵ The differences in MHC‐II between donors of MSCs and responder PBMCs were verified in parallel studies in which PBMCs from the donor MSCs served as stimulators (not shown). This verification was significant since reduced proliferation cannot be attributed to similarities in MHC‐II. Similar proliferation was observed with 4‐day‐induced MSCs (Figure 3, diagonal bar). PBMC proliferations were reduced at 6‐ and 12‐day‐induced MSCs (Figure 3). Interestingly, day 8‐induced MSCs showed enhanced proliferation, which was significantly (p < 0.05) higher than 6‐ and 12‐day‐induced stimulator MSCs. In summary, excluding day 8‐induced MSCs, there were reduced allogeneic responses by MSCs induced for more than 4 days.

Figure 3.

Allogeneic effects of MSCs, uninduced (0) or induced for different times. Stimulator MSCs and the derived neurons were cultured in the presence of allogeneic responder PBMCs for 72 hours with 16‐hour pulse with TdR. The results are presented as the mean DPM ± SD, n= 6. Each experiment was done with MSCs and PBMCs from different donors. *p < 0.05 versus cultures with 6‐ and 12‐day‐induced cells.

Expression of MHC‐II in MSCs placed in one‐way MLR cultures

MHC‐II expression is gradually reduced in MSCs subjected to neuronal induction (Figure 1B). Since 8‐day‐induced MSCs mediated increased proliferation in one‐way MLR, we next asked whether this could be attributed to the reexpression of MHC‐II in the one‐way MLR. We therefore removed the nonadherent cells in one‐way MLR cultures and then studied MHC‐II expression in the adherent cells (green fluorescence). To avoid forceful washing to eliminate all of the nonadherent cells, we also labeled with CD45 (red fluorescence) since activated T cells and B cells express MHC‐II. Cells were identified by nuclei labeling with DAPI (blue). As expected, MHC‐II was detected in PBMC alone, uninduced MSCs, and day 4‐induced cells (Figure 4, green). MSCs induced for 6, 8, and 12 days show dim to negative green fluorescence, indicating minimal MHC‐II expression. Parallel studies with PE‐CD14 for macrophages showed no labeling (not shown). This finding indicates that the green fluorescence is attributed to MSCs. In summary, the results show no significant reexpression of MHC‐II at day 8‐induced MSCs.

Figure 4.

MHC‐II in MSCs (uninduced and induced) with allogeneic PBMCs. Cultures were established as for Figure 2, except that the MSCs were induced on glass slides. After 72 hours, the nonadherent cells were washed and the adherent cells were labeled by two‐color immunofluorescence with FITC‐anti‐MHC‐II and PE‐anti‐CD45. The cells were counterstained for nuclei localization with DAPI. The studies represent three different experiments, each done with MSCs and PBMCs from different donors.

Role of the neurotransmitter SP in one‐way MLR with day 8‐induced MSCs

The increase in PBMCs proliferation in one‐way MLR with 8‐day‐induced MSCs could not be explained by the reexpression of MHC‐II (Figure 4). We therefore asked whether this could be explained by the release of neurotransmitters from the developing MSCs. This question is plausible because MSC‐derived neurons have been shown to be capable of expressing several neurotransmitters with proinflammatory roles. ¹⁹ , ²⁸ , ²⁹ One‐way MLR were performed in the presence or absence of the calcium chelator, BAPTA. This inhibited vesicular neurotransmitter release from the induced MSCs and, therefore, made the neurotransmitters unavailable for immune cell stimulation. ³⁰ The results showed no change in PBMC proliferation for uninduced and day 4‐induced MSCs (Figure 5A). However, there was significant reduction in PBMC proliferation at day 8 when BAPTA was present in the cultures (Figure 5A). Interestingly, we observed significantly (p < 0.05) increased proliferation for 12‐day‐induced MSCs (Figure 5A). In summary, the results indicated a role for neurotransmitters as mediators of PBMC proliferation in the one‐way MLR.

Figure 5.

Role of the neurotransmitter SP in the proliferation of PBMCs in contact with MSCs (uninduced (0) or induced for different times). (A) The studies were established as for Figure 3, except that the neurons were pretreated with 25 μM of BAPTA or with vehicle for 30 minutes. The results are presented as the mean DPM ± SD, n= 4. *p< 0.05 versus cultures with day 8‐induced cells with vehicle; **p< 0.05 versus cultures with day 12‐induced cells with vehicle. (B) One‐way MLR was established as for Figure 3 except that the cultures were done in the presence or absence of 10 nM NK1 antagonist, CP‐99,994. MSCs induced for 6 days (D6) served as control for nonspecific effects of the antagonist. The results are presented as the mean DPM ± SD, n= 5. *p< 0.05 versus 8 day cultures with vehicle alone. (C) The media from “A” was collected after 24 hours and then studied for SP levels by ELISA. The results are presented as the mean ± SD SP levels (pg/mL, n= 5).

NK1 receptor in the proliferation of PBMC in one‐way MLR with day 8‐induced MSCs

The release of neurotransmitter by 8‐day‐induced MSCs appears to have a role in the increased proliferation of PBMCs (Figure 5A). This section investigated the neurotransmitter SP mainly due to its role as a mediator of PBMC proliferation. ³¹ , ³² In addition, 8‐day‐induced MSCs express the SP gene, Tac1, at the mRNA levels with a low level of SP translation. ¹² , ¹⁹ Increases in Tac1 mRNA translation could occur in the presence of proinflammatory cytokines. ²² , ³³ Thus, it is possible that Tac1 mRNA might be translated if cytokines are produced by the stimulator PBMCs in the one‐way MLR. To determine a role for SP, we repeated the one‐way MLR in the presence or absence of the receptor antagonist, CP‐99,994. This antagonist is specific for the high‐affinity NK1, which has been detected on MSC‐derived neurons and has been shown to be involved in SP reuptake. ¹⁹ , ²² Nonspecific effects of the antagonist were examined in parallel assays with 6‐day‐induced MSCs. The results show significant (p < 0.05) reduction in PBMC proliferation in the presence of the antagonist for assays with 8‐day‐induced MSCs (Figure 5B). Similar changes were not observed in vehicle or in cultures with 6‐day‐induced MSCs (Figure 5B). In summary, the results indicate a role for NK1 for PBMCs proliferation in one‐way MLR with 8‐day‐induced MSCs.

Production of SP by MSC‐derived neurons

Studies with NK1 antagonist indicated a role for this receptor in PBMC proliferation of one‐way MLR (Figure 5B). In addition, studies with BAPTA indicated the involvement of released neurotransmitter(s) (Figure 5A). Day 8‐induced neurons are immature with respect to their electrophysiological properties. ¹² , ¹⁹ Furthermore, we have irradiated the induced MSCs before placing them as stimulators in the one‐way MLR. Thus, it is likely that the relevant NK1 receptors are present on the responder PBMCs of the one‐way MLR cultures. ³⁴ To this end, we have studied the culture media for SP levels and found significantly (p < 0.05) higher levels in cultures with day 8‐induced MSCs, as compared to the other time points (Figure 5C). In addition, the data also showed significant (p < 0.05) reduction of SP in cultures with BAPTA, suggesting the induced cells as the source of SP (Figure 5C).

miRNA levels in MSCs, induced and uninduced from MLR

We have previously reported reduced translation of Tac1 mRNA in induced MSCs. ³³ Thus, we asked whether during the MLR reaction, the Tac1‐specific miRNAs are decreased in 8‐day‐induced MSCs from MLR reactions. qPCR was performed with total RNA from MSCs isolated in MLR for miR‐206 and miR‐130a, previously reported to blunt Tac1 mRNA translation. ³³ The results showed significant (p < 0.05) decreases in the levels of both miRNAs for 8‐day‐induced MSCs as compared to uninduced cells (Figure 6A and 6B). In summary, the results show decreases in miRNA130a and miRNA206 in stimulator MSCs that correlated with increased SP levels.

Figure 6.

miRNA levels in 8‐day‐induced MSCs from MLR. Reactions were established as for Figure 3. At 72 hours, total RNA was isolated from the adherent/CD45 negative population and then subjected to qPCR for miR206 (A) and −130a (B). The results for D0 were normalized to 1 and the results for D8 and D12 were presented as the mean percentage change ± SD, n= 4.

Cytokines, CGRP, and VIP in induced and uninduced MSCs

Since 12‐day induction resulted in functional neurons, ¹² , ¹⁹ it is possible that the release of other neurotransmitters might be responsible for the reduced cell proliferation in MLR with 12‐day‐induced MSCs (Figure 5A). We focused on CGRP and VIP, both of which have been shown to mediate antiinflammatory responses. ³⁵ , ³⁶ Western blots were performed for VIP and CGRP with whole cell extracts obtained from MSC, 8‐ and 12‐day induced (8 and 12) and uninduced (0). The results, representative of three experiments, showed a strong band for VIP in the uninduced cells, which was decreased upon induction (Figure 7A and 7B). In contrast, strong bands were observed for CGRP following induction.The band for CGRP was dim in 8‐day‐induced MSCs (Figure 7A and 7C). Since this neurotransmitter has been linked to immune suppression, ³⁶ a role for SP was further supported (Figure 5B and 5C). However, SP can induce the production of cytokines with proinflammatory properties. ³⁷ We therefore studied cytokine production in MLR with 8‐day‐induced MSCs and compared with the following: MLR with 12‐day‐induced and uninduced MSCs. The data are presented for proinflammatory cytokines, IL‐2, TNFα, IFNγ, IL‐1α, and TGF−b and the anti‐inflammatory cytokine IL‐10. The levels of IL‐2 and IL‐10 were similar for uninduced (D0) and 8‐day‐induced (D8) (Table 1 and Figure 8). However, the levels of the other cytokines were significantly increased in MLR with D8 MSCs (Table 1). In summary, the results showed significant increase in proinflammatory cytokines in MLR with D8‐induced MSCs, but not in cultures with D12‐induced MSCs.

Figure 7.

Neuropeptide proteins in uninduced (DO) and induced (D8 and D12) MSCs. (A) Whole cell extracts from uninduced and induced MSCs were analyzed by Western blots with anti‐VIP and anti‐CGRP. Normalizations were performed with anti‐β‐actin. Representative blot is shown for three experiments. The band densities for VIP (B) and CGRP (C) are shown as mean ± SD.

Table 1.

Baseline cytokine production in mscs and PBmcs. Cell‐free media from uninduced MSCs and unstimulated PBMCs were analyzed for cytokine production using protein arrays. Each experiment was performed four times, each with cells from a different donor. The densitometric scans were normalized to 10, and the normalized alues are presented as the mean ± SD.

Cytokines	MSCs	PBMCs
IL‐2	ND	ND
TNFα	ND	ND
IL‐1α	ND	ND
IFNγ	ND	ND
TGF‐β	1.7 ± 0.1	ND
IL‐10	1.4 ± 0.2	ND

ND: None detected

Figure 8.

Cytokine produce in MLRs. Cell‐free media from MLR with uninduced (day 0) and induced MSCs (day 8 and day 12) were analyzed on cytokine arrays. Parallel studies with media from PBMC or MSCs cultured alone are shown in Table 1. Each experiment was performed four times, each with cells from a different donor. The densitometric scans were normalized to 10, and the normalized values are presented as the mean ± SD.

Expression of MHC‐I and MHC‐II in MSC‐derived neurons

MHC‐II expression was developmentally decreased in the MSCs subjected to neuronal induction (Figure 1B). In addition, we did not detect MHC‐II in the stimulator 12‐day‐induced MSCs from one‐way MLR cultures (Figure 4). This could be attributed to antagonistic and/or synergistic effects of factors produced in the one‐way MLR. Thus, to determine if the induced MSCs are capable of reexpressing MHC‐II, we performed cultures in the absence of PBMC, but instead, stimulated the induced cells for 16 hours with 10 U/mL IFNγ. This concentration of IFNγ was selected since it maintains the expression of MHC‐II on MSCs. ¹³ Western blots showed reduced expression in 12‐day induction, in the absence of IFNγ (Figure 9A and 9C). However, at all time periods, MHC‐II was reexpressed in the presence of IFNγ (Figure 9A and 9C). We next determined whether IFNγ mediated changes in MHC‐I expression. We therefore analyzed the extracts from Figure 9A for MHC‐I by Western blots. The results showed no change in MHC‐I expression (Figure 9B and 9D). In summary, the results show MSC‐derived neurons are capable of reexpressing MHC‐II, although there are no changes in MHC‐I.

Figure 9.

MHC‐I and ‐II in IFγ‐stimulated MSCs, uninduced MSCs (DO) or induced for different times (D4, D8, D12). Cells were stimulated with 10 U/mL of IFNγ (+). After 12 hours, membrane extracts were isolated and then subjected to Western blots for MHC‐II (A) and MHC ‐I (B) with the respective antibodies. The band densities for MHC‐I and MHC ‐II are presented in (C) and (D). Unstimulated cells served as controls (−). Membrane was stripped and reprobed with anti‐β‐actin (n= 4).

Discussion

The pluripotency of MSCs and their unique immune properties make them leading candidate stem cells for regenerative medicine. A major point in the argument for MSCs to be transplanted across allogeneic barriers is based on the immune‐suppressive properties of these stem cells. ³ The vast literature eliminates doubts on the ability of MSCs to differentiate and transdifferentiate into multiple cell types. This potential of MSCs brings up two major points. Firstly, how would these cells interact with the microenvironment, and secondly, would the specialized stem cells reexpress MHC‐II to cause rejection of the implanted cells? An appealing property of MSCs is the minimization of ethical issues as compared to embryonic and fetal stem cells.

This report took a close look at the translational potential of MSC‐derived neurons, by in vitro methods. Other studies on microenvironmental influence by cytokines are not mutually exclusive of the presented studies. ²² The ability of IFNγ to mediate the reexpression of MHC‐II, even in the mature neurons (Figure 9), indicates that in translational applications, the surrounding tissue would be relevant to the responses of the implanted cells. Despite the observed reexpression of MHC‐II, this finding does not propose to eliminate allogeneic MSCs for neural repair. Controlled studies are needed to determine how chimerism could be maintained, similar for patients who have undergone hematopoietic stem cell transplantation for decades. Also, since these studies were done by in vitro methods, future in vivo studies would determine if synergism and/or antagonism among tissue factors might circumvent the reexpression of MHC‐II.

The neurotransmitter SP is likely to be relevant to the proliferation of PBMCs at day 8. The question is why we did not observe similar findings for 12‐day‐induced MSCs (Figure 3). MSCs that have been subjected to the induction protocol could produce various neurotransmitters, including those with immune‐suppressive effects. It is possible that by day 12, other neurotransmitters are released to negatively affect the effects of SP. We did not observe high levels of SP at day 12 induction as compared to day 8‐induced MSCs (Figure 5C). Since SP is an unstable neurotransmitter, it is possible that it is degraded after day 8 by endopeptidases, or could be degraded intracellularly. ³⁸ , ³⁹ We observed increased production of the immune‐suppressive neurotransmitter CGRP (Figure 7). ³⁶ Despite the increased bands for CGRP, this study did not address a cause‐effect relationship between this neurotransmitter and the main findings of the report.

The main point in this study is the peak proliferation in MLR with 8‐day‐induced MSCs (3, 5). MSC‐derived neurons express the Tac1 mRNA, but show minimum translation due to specific miRNAs. ³³ We now show that the responsive PBMCs might be involved in degrading Tac1‐specific miRNA (Figure 6). At present, it is unclear if the decreased levels of miRNAs occur indirectly via cytokines. Although NK1 antagonists and BAPTA suggest a strong role for SP, this neurotransmitter appears to be acting via the production of other proinflammatory cytokines (Table 1 and Figure 8). An intriguing finding is the high production of IFNγ in MLR stimulated with 8‐day‐induced MSCs (Figure 8). This correlated with a band for MHC‐II in day 8‐induced MSCs (Figure 9). Since IFNγ was decreased in MLR with 12‐day‐induced MSCs, we speculate that this might be responsible for the decrease in MHC‐II in the neurons (Figure 9A), and perhaps increases or decreases in cytokine productions (8, 9). Despite the downregulation of MHC‐II in the induced cells, IFNγ can mediate its reexpression (Figure 9A). However, similar changes were not observed for MHC‐I (Figure 9B).

CIITA is expected to be present within the cytosol during MHC‐II decrease (1, 2). However, this finding is interesting since it could be a potential target for silencing MHC‐II in transplanted allogeneic MSCs. It could be argued that genetic manipulation might be unnecessary when it is relatively easy to harvest and expand MSCs with bone marrow aspirates. This is true for patients who can afford the delay linked to harvesting bone marrow, expanding MSCs, and characterizing for transplantation. In cases such as traumatic brain injury and spinal cord injury, this waiting period might not be an option. In these cases, “off the shelf” MSCs will require investigations into various strategies to prevent them from causing harm. Another case where autologous MSCs might not be an option would be in genetically linked diseases where the candidate genes might be reexpressed after MSCs have transitioned to the specialized cells.

Another point that we cannot ignore is the possibility of minor stimulatory effects by MHC‐I. It would be interesting to determine if this could be circumvented by a tolerogenic mechanism where the host would not reject the MHC‐I expressing new cells. Also, the specialized cells would be derived from stem cells, which could eventually express an oncogene or suppress a tumor suppressor gene. This brings up the question of whether natural killer cells would respond to the different MHC‐I. The immune effects of transdifferentiated neurons are paramount for the translation of stem cells to patients. The science has proven that adult stem cells can form different specialized cells by in vitro methods. Thus, the in vivo relevance is still questionable. The next step is to be able to implant them safely. Studies such as this article will contribute to stem cell translation to patients.