Neurons Derived From Human Mesenchymal Stem Cells Show Synaptic Transmission and Can Be Induced to Produce the Neurotransmitter Substance P
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《干细胞学杂志》
a Department of Medicine, Hematology/Oncology and Graduate School of Biomedical Science-Interdisciplinary Program and
b Department of Pharmacology and Physiology, Anesthesiology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA;
c Department of Clinical Laboratory Science, Korea University, Seoul, Korea
Key Words. Mesenchymal stem cells ? Transdifferentiation ? Preprotachykinin-I ? Neurons
Correspondence: Pranela Rameshwar, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103, USA. Telephone: 973-972-0625; fax: 973-972-8854; e-mail: rameshwa@umdnj.edu
ABSTRACT
Research studies on adult and embryonic stem cells (ESCs) have undergone enormous advances during the past few years. The experimental evidence shows that both stem cells could have future benefits in the area of regenerative medicine. Adult stem cells have been shown to have potential benefits for such diseases or conditions as diabetes mellitus, liver disease, cardiac dysfunction, Alzheimer’s disease, Parkinson’s disease, spinal cord injuries, bone defects, and genetic abnormalities . Furthermore, adult stem cells have been extensively studied for repair of orthopedic conditions, such as bone, cartilage, and tendon defects .
ESCs are considered the prototype stem cells because of their innate ability to differentiate into all possible cells and are therefore invaluable sources for tissue repair . Despite the remarkable potential of ESCs in medicine, the obvious limit is the need for a safe match with respect to the major histocompatibility complex class II . ESCs could become functionally unstable when placed in an in vivo microenvironment and develop into tumors . Another important consideration for ESCs is the stage of development. These cells might be in transit to committed cells and would therefore, express various developmental genes. This molecular change might not be evident, because the ESCs might not show phenotypic changes. Thus, it is conceivable that cells generated from ESCs could be dysfunctional if the originating stem cells are already committed to form cells of another tissue. Given these arguments, clinical application of ESCs would require robust examination of gene expressions before the generation of different cell types.
Clinical application of hematopoietic stem cells (HSCs) is controversial. Some reports show evidence of transdifferentiation by HSCs . Others report that transdifferentiation of HSCs could be mistaken by cell fusion between HSCs and cells of other tissues . An issue that is mostly overlooked with respect to HSCs is the low efficiency that one could achieve in their expansion by current in vitro methods. Thus, to acquire sufficient HSCs for clinical use would require invasive procedures upon the donors. Another issue with HSCs is that a population of HSCs that is deemed stem cells by phenotypic analyses is generally heterogeneous and could include cells that are committed towards a particular lineage . Future application of HSCs in repair medicine requires further research with the appropriate cell subset. Given an ideal situation where the candidate HSC is identified, there are ethical issues on the amount of bone marrow (BM) aspirates that should be taken from a donor. Presently, the literature has opened potential avenues for all types of stem cells. The most efficient stem cell or cells with least ethical issues will be determined by future research studies.
Accordingly, alternative use of adult stem cells has been examined . Mesenchymal stem cells (MSCs) show promise among the adult stem cells. MSCs are major adult BM stem cells with multilineage potential . Theoretically, MSCs can be used across allogeneic barrier because of their unique immune property . This property of MSCs is demonstrated by the cell’s ability to facilitate BM transplantation. . Furthermore, MSCs are easily obtained from adult BM and can be expanded by simple in vitro procedures . MSCs have been shown to transdifferentiate into cells of other germ layers .
The generation of MSCs into neurons has been studied . However, for the most part, these studies characterized the transdifferentiation of MSCs based on morphology, phenotypic changes, and action potential . As far as we are aware, synaptic transmission has not been reported for neurons derived from MSCs. Cells similar to MSCs have been shown to survive in the brain . In this report, we isolated MSCs from human BM aspirate and generated functional neurons as indicated by phenotype and electrophysiology. Synaptic transmission, as evident by immunofluorescence for synaptophysin, was supported by the presence of synaptic currents.
Administration or implantation of cultured neurons in a damaged tissue in vivo would be influenced by the microenvironment. Thus, the question is whether a neuron could be influenced to express a particular neurotransmitter. This is a relevant question, because microenvironmental changes would vary depending on the type of deregulation or injury. This study focused on the preprotachykinin-I gene (PPT-I), which encodes the neurotransmitter substance P (SP). Regulation of the PPT-I gene has been studied in the context of inflammatory mediators that are presumed to be at the site of neural injury. Interleukin (IL)-1 was selected mostly because of its role in neural function , its ability to induce the production of SP , and its signature as a proinflammatory mediator .
MATERIALS AND METHODS
Transdifferentiation of MSCs to Neuron-Like Cells
MSCs treated with RA were first examined microscopically at x 400 magnification. The purpose was to observe morphological changes as the cells were exposed to a known differentiating agent. Compared with the symmetrical morphology of untreated MSCs (Fig. 1, upper left panel), MSCs treated for 1 day with RA showed a transition to cells with asymmetrical morphology (Fig. 1, upper right panel). This change progressed to day 4, when the RA-treated MSCs showed neuron-like structures (Fig. 1, lower left panel). By day 7, the treated cells demonstrated structures of cell bodies with long thin processes and growth cones (Fig. 1, lower right panel).
Figure 1. Morphology of MSCs after treatment with RA. MSCs were treated with RA for different times and then examined by light microscopy. Shown are untreated MSCs (upper left panel) and RA-treated cells for 1, 4, and 7 days. Abbreviations: MSC, mesenchymal stem cell; RA, retinoic acid.
We next determined whether the RA-treated MSCs express neuron-specific proteins. Cells were labeled for MAP2, which mostly labels the cell bodies and dendrites, neurofilament to stain for cell bodies, and axons and nestin for changes in morphology to neuronal cells. Cells were also studied for synaptophysin, which is an indicator of synaptic regions. Labeling with anti-MAP2 (FITC) and anti-synaptophysin (PE) were done at different times after RA treatment. Figure 2 shows pictures of individually labeled and overlaid MAP2 and synaptophysin (left columns).
Figure 2. Immunofluorescence for neuronal markers. (A): MSCs were treated with RA, and at days 6, 9, and 12, cells were double labeled for MAP2 (fluorescein isothiocyanate) and synaptophysin (phycoerythrin). Overlays of MAP2 and synaptophysin staining are shown at the extreme right. Shown are 10 experiments, each performed with MSCs from a different donor. (B): MSCs were treated with RA, and at day 6, cells were stained for neurofilament (68 kDa) or nestin. Shown are five different experiments, each with a different donor. Abbreviations: MSC, mesenchymal stem cell; RA, retinoic acid.
Untreated MSCs stained dim with MAP2 but negative for synaptophysin (Fig. 2, top panels). In contrast, RA-treated cells stained bright for MAP2 at each time point shown in Figure 2. Cells stained for 6-day RA treatment showed predominance of MAP2 and slight fluorescence for synaptophysin (Fig. 2). At day 9, RA-treated cells showed brighter fluorescence for synaptophsin. This pattern of synaptophysin increased at up to day 12 of treatment. Figure 2A represents 10 different experiments, each with MSCs from a different BM donor.
Figure 2B shows representative staining for neurofilament and nestin. All cells treated with RA were negative for GFAP (not shown). The percentage of neurons developed by RA treatment were 80 ± 10 (n = 100) by immunostaining for neurofilament, MAP2, and nestin. However, when the RA-treated cells were analyzed by electrophysiology, 100% showed action potential (n = 25). Of note is that transfer of MSCs from propagation media (DMEM) to DMEM/F12 does not increase the percentages of neuron formation but enhanced the formation of nestin-positive cells by 2–3 days. The results show that MSCs treated with RA coexpress MAP2 and synaptophysin beginning at day 6 of RA treatment.
Electrophysiology in RA-Treated MSCs
To determine whether the RA-treated MSCs show functions consistent with native neurons, we studied the cells’ electrophysiological properties using whole-cell patch clamp technique. First, record was made of action potentials under current clamp condition. As illustrated in Figure 3A, the cell fired spontaneously and regularly with a frequency of 20 s–1. The amplitude, measured from the resting membrane potential of –50 mV, was 63.4 ± 17.4 mV. The half-width and rise time of the action potentials were 19.25 ± 0.73 and 3.39 ± 0.11 ms (n = 768 events), respectively (Fig. 3D). To determine whether the RA-treated cells can communicate with each other, we next measured the postsynaptic currents under voltage clamp condition. Figure 3B illustrates a segment of such trace recorded from a cell treated with RA for 6 days. The frequency, amplitude, half rise time, and decay time of the postsynaptic currents of this cell were 2.49 s–1, 29.9 ± 1.3 pA, 12.96 ± 0.89 ms, and 36.5 ± 1.0 ms (n = 171 events), respectively. Figure 3C illustrates representative postsynaptic currents recorded from a cell in culture for 15 days. The frequency, amplitude, half rise time, and decay time of the postsynaptic currents of this cell were 1.24 s–1, 6.1 ± 0.2 pA, 1.84 ± 0.13 ms, and 31.3 ± 1.3 ms (n = 99 events), respectively (Fig. 3D). Kinetic analysis indicated that although the decay of the postsynaptic currents of the cell in culture for 6 days could be fitted by a single exponential function, the decay of the postsynaptic currents of the cell in culture for 15 days required two exponentials.
Figure 3. Electrophysiological properties of retinoic acid-treated mesenchymal stem cells. (A): Ongoing discharges recorded from a cell in culture for 4 days under current clamp condition. The resting membrane potential is at –50 mV. An accelerated trace is shown below. (B): Postsynaptic currents (PSCs) recorded at a holding potential of –60 mV from a cell in culture for 6 days. The trace below shows the decay of the PSC could be fitted by a single exponential. (C): PSCs recorded at a holding potential of –60 mV from a cell in culture for 15 days. The trace below in extended time scale shows the decay of the PSC could be fitted by two exponentials and has a time constant of 1 = 3.7 and 2 = 32.4 ms. The amplitudes were A1 = 3 pA and A2 = 24 pA. (D): Histogram of half-rise times and decay times of PSCs measured from cells in culture of 6 and 15 days.
Induction of SP in RA-Treated MSCs
This section describes studies to determine whether the neurons formed from MSCs could be induced to express a particular neurotransmitter. We selected IL-1 and the neurotransmitter gene PPT-I, because the regulation of PPT-I by inflammatory mediators such as IL-1 has been studied . Furthermore, the receptor for IL-1 has been reported in neurons . In addition, IL-1 can induce the production of SP, which is the major peptide derived from the PPT-I gene .
MSCs, treated with RA for 6 days, were stimulated with 10 ng/ml of IL-1 for 16 hours, and the cells were studied for SP production by immunofluorescence using anti-SP. The treated cells were also labeled for synaptovesicle 2, because it would be of interest to determine whether SP is stored in synaptic vesicle. The studies used MSCs exposed to RA for 6 days, because this time frame also showed functional synaptic activity (Fig. 3). RA-treated MSCs were labeled with anti-SP (FITC; Fig. 4, left column) and anti-synaptovesicle (PE; Fig. 4, middle panel). The two labels were overlaid to get insights on the location of SP. Because the anti-SP was diluted in 1% HSA and the immunofluorescence used indirect immunofluorescence, secondary antibody specificity was studied in parallel labeling with 1% HSA in lieu of anti-SP.
Figure 4. Immunohistochemistry for SP in MSC-derived neurons stimulated with IL-1. MSCs were treated with RA for 10 days and then stimulated with 25 ng/ml of IL-1 in the presence or absence of 4 μg/ml IL-RA. Control neurons contained vehicle for IL-1 (1% HSA). After 16 hours of stimulation, cells were labeled for SP by immunofluorescence. Shown are six experiments, each performed with MSCs from a different donor. Abbreviations: HSA, human serum albumin; IL, interleukin; IL-RA, IL-1 receptor antagonist; MSC, mesenchymal stem cell; RA, retinoic acid; SP, substance P.
Representations of six experiments, each performed with a different donor, are shown in Figure 4. The results show bright fluorescence intensity for SP in cells stimulated with IL-1 (Fig. 4, middle row). Fluorescence intensities were significantly reduced when the cells were costimulated with IL-1 RA (Fig. 4, bottom row). This indicates that the effects of IL-1 were specific. Cells labeled with 1% HSA showed minimal fluorescence, which was subsequently used as background fluorescence to compare other labeling studies (Fig. 4, top row). The results show that IL-1 can induce the production of SP in neurons generated from RA-treated MSCs.
Expression of ?-PPT-I in RA-Treated MSCs
Because dim fluorescence was observed for SP in studies with unstimulated MSCs treated with RA (not shown), we surmised that the gene from which SP is produced, PPT-I, might be expressed with low level of translation. We therefore asked whether the PPT-I gene was expressed in RA-treated MSCs by in situ hybridization with a cocktail of three oligomers (Table 1). Studies with neurons from three different BM donors are shown in Figure 5. Bright fluorescence was observed in the cell bodies of neurons (Fig. 5, right panels). Neurons labeled with sense oligonucleotides showed no fluorescence (not shown). The results indicate that the PPT-I gene is expressed in untreated MSCs differentiated with RA.
Figure 5. Expression of PPT-I in RA-treated MSCs. In situ hybridization for ?-PPT-I was performed with MSCs treated with RA for 10 days. Studies are shown for labeling with neurons generated from three different donors. Abbreviations: MSC, mesenchymal stem cell; PPT-I, preprotachykinin-I; RA, retinoic acid.
DISCUSSION
MSCs can be induced, via RA treatment, to become functional neurons that express neuron-specific phenotypic markers and exhibit synaptic properties similar to those of native neurons. The MSC-derived neurons express the tachykinin gene PPT-1 and can be induced to produce SP by IL-1 stimulation.
ACKNOWLEDGMENTS
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b Department of Pharmacology and Physiology, Anesthesiology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA;
c Department of Clinical Laboratory Science, Korea University, Seoul, Korea
Key Words. Mesenchymal stem cells ? Transdifferentiation ? Preprotachykinin-I ? Neurons
Correspondence: Pranela Rameshwar, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103, USA. Telephone: 973-972-0625; fax: 973-972-8854; e-mail: rameshwa@umdnj.edu
ABSTRACT
Research studies on adult and embryonic stem cells (ESCs) have undergone enormous advances during the past few years. The experimental evidence shows that both stem cells could have future benefits in the area of regenerative medicine. Adult stem cells have been shown to have potential benefits for such diseases or conditions as diabetes mellitus, liver disease, cardiac dysfunction, Alzheimer’s disease, Parkinson’s disease, spinal cord injuries, bone defects, and genetic abnormalities . Furthermore, adult stem cells have been extensively studied for repair of orthopedic conditions, such as bone, cartilage, and tendon defects .
ESCs are considered the prototype stem cells because of their innate ability to differentiate into all possible cells and are therefore invaluable sources for tissue repair . Despite the remarkable potential of ESCs in medicine, the obvious limit is the need for a safe match with respect to the major histocompatibility complex class II . ESCs could become functionally unstable when placed in an in vivo microenvironment and develop into tumors . Another important consideration for ESCs is the stage of development. These cells might be in transit to committed cells and would therefore, express various developmental genes. This molecular change might not be evident, because the ESCs might not show phenotypic changes. Thus, it is conceivable that cells generated from ESCs could be dysfunctional if the originating stem cells are already committed to form cells of another tissue. Given these arguments, clinical application of ESCs would require robust examination of gene expressions before the generation of different cell types.
Clinical application of hematopoietic stem cells (HSCs) is controversial. Some reports show evidence of transdifferentiation by HSCs . Others report that transdifferentiation of HSCs could be mistaken by cell fusion between HSCs and cells of other tissues . An issue that is mostly overlooked with respect to HSCs is the low efficiency that one could achieve in their expansion by current in vitro methods. Thus, to acquire sufficient HSCs for clinical use would require invasive procedures upon the donors. Another issue with HSCs is that a population of HSCs that is deemed stem cells by phenotypic analyses is generally heterogeneous and could include cells that are committed towards a particular lineage . Future application of HSCs in repair medicine requires further research with the appropriate cell subset. Given an ideal situation where the candidate HSC is identified, there are ethical issues on the amount of bone marrow (BM) aspirates that should be taken from a donor. Presently, the literature has opened potential avenues for all types of stem cells. The most efficient stem cell or cells with least ethical issues will be determined by future research studies.
Accordingly, alternative use of adult stem cells has been examined . Mesenchymal stem cells (MSCs) show promise among the adult stem cells. MSCs are major adult BM stem cells with multilineage potential . Theoretically, MSCs can be used across allogeneic barrier because of their unique immune property . This property of MSCs is demonstrated by the cell’s ability to facilitate BM transplantation. . Furthermore, MSCs are easily obtained from adult BM and can be expanded by simple in vitro procedures . MSCs have been shown to transdifferentiate into cells of other germ layers .
The generation of MSCs into neurons has been studied . However, for the most part, these studies characterized the transdifferentiation of MSCs based on morphology, phenotypic changes, and action potential . As far as we are aware, synaptic transmission has not been reported for neurons derived from MSCs. Cells similar to MSCs have been shown to survive in the brain . In this report, we isolated MSCs from human BM aspirate and generated functional neurons as indicated by phenotype and electrophysiology. Synaptic transmission, as evident by immunofluorescence for synaptophysin, was supported by the presence of synaptic currents.
Administration or implantation of cultured neurons in a damaged tissue in vivo would be influenced by the microenvironment. Thus, the question is whether a neuron could be influenced to express a particular neurotransmitter. This is a relevant question, because microenvironmental changes would vary depending on the type of deregulation or injury. This study focused on the preprotachykinin-I gene (PPT-I), which encodes the neurotransmitter substance P (SP). Regulation of the PPT-I gene has been studied in the context of inflammatory mediators that are presumed to be at the site of neural injury. Interleukin (IL)-1 was selected mostly because of its role in neural function , its ability to induce the production of SP , and its signature as a proinflammatory mediator .
MATERIALS AND METHODS
Transdifferentiation of MSCs to Neuron-Like Cells
MSCs treated with RA were first examined microscopically at x 400 magnification. The purpose was to observe morphological changes as the cells were exposed to a known differentiating agent. Compared with the symmetrical morphology of untreated MSCs (Fig. 1, upper left panel), MSCs treated for 1 day with RA showed a transition to cells with asymmetrical morphology (Fig. 1, upper right panel). This change progressed to day 4, when the RA-treated MSCs showed neuron-like structures (Fig. 1, lower left panel). By day 7, the treated cells demonstrated structures of cell bodies with long thin processes and growth cones (Fig. 1, lower right panel).
Figure 1. Morphology of MSCs after treatment with RA. MSCs were treated with RA for different times and then examined by light microscopy. Shown are untreated MSCs (upper left panel) and RA-treated cells for 1, 4, and 7 days. Abbreviations: MSC, mesenchymal stem cell; RA, retinoic acid.
We next determined whether the RA-treated MSCs express neuron-specific proteins. Cells were labeled for MAP2, which mostly labels the cell bodies and dendrites, neurofilament to stain for cell bodies, and axons and nestin for changes in morphology to neuronal cells. Cells were also studied for synaptophysin, which is an indicator of synaptic regions. Labeling with anti-MAP2 (FITC) and anti-synaptophysin (PE) were done at different times after RA treatment. Figure 2 shows pictures of individually labeled and overlaid MAP2 and synaptophysin (left columns).
Figure 2. Immunofluorescence for neuronal markers. (A): MSCs were treated with RA, and at days 6, 9, and 12, cells were double labeled for MAP2 (fluorescein isothiocyanate) and synaptophysin (phycoerythrin). Overlays of MAP2 and synaptophysin staining are shown at the extreme right. Shown are 10 experiments, each performed with MSCs from a different donor. (B): MSCs were treated with RA, and at day 6, cells were stained for neurofilament (68 kDa) or nestin. Shown are five different experiments, each with a different donor. Abbreviations: MSC, mesenchymal stem cell; RA, retinoic acid.
Untreated MSCs stained dim with MAP2 but negative for synaptophysin (Fig. 2, top panels). In contrast, RA-treated cells stained bright for MAP2 at each time point shown in Figure 2. Cells stained for 6-day RA treatment showed predominance of MAP2 and slight fluorescence for synaptophysin (Fig. 2). At day 9, RA-treated cells showed brighter fluorescence for synaptophsin. This pattern of synaptophysin increased at up to day 12 of treatment. Figure 2A represents 10 different experiments, each with MSCs from a different BM donor.
Figure 2B shows representative staining for neurofilament and nestin. All cells treated with RA were negative for GFAP (not shown). The percentage of neurons developed by RA treatment were 80 ± 10 (n = 100) by immunostaining for neurofilament, MAP2, and nestin. However, when the RA-treated cells were analyzed by electrophysiology, 100% showed action potential (n = 25). Of note is that transfer of MSCs from propagation media (DMEM) to DMEM/F12 does not increase the percentages of neuron formation but enhanced the formation of nestin-positive cells by 2–3 days. The results show that MSCs treated with RA coexpress MAP2 and synaptophysin beginning at day 6 of RA treatment.
Electrophysiology in RA-Treated MSCs
To determine whether the RA-treated MSCs show functions consistent with native neurons, we studied the cells’ electrophysiological properties using whole-cell patch clamp technique. First, record was made of action potentials under current clamp condition. As illustrated in Figure 3A, the cell fired spontaneously and regularly with a frequency of 20 s–1. The amplitude, measured from the resting membrane potential of –50 mV, was 63.4 ± 17.4 mV. The half-width and rise time of the action potentials were 19.25 ± 0.73 and 3.39 ± 0.11 ms (n = 768 events), respectively (Fig. 3D). To determine whether the RA-treated cells can communicate with each other, we next measured the postsynaptic currents under voltage clamp condition. Figure 3B illustrates a segment of such trace recorded from a cell treated with RA for 6 days. The frequency, amplitude, half rise time, and decay time of the postsynaptic currents of this cell were 2.49 s–1, 29.9 ± 1.3 pA, 12.96 ± 0.89 ms, and 36.5 ± 1.0 ms (n = 171 events), respectively. Figure 3C illustrates representative postsynaptic currents recorded from a cell in culture for 15 days. The frequency, amplitude, half rise time, and decay time of the postsynaptic currents of this cell were 1.24 s–1, 6.1 ± 0.2 pA, 1.84 ± 0.13 ms, and 31.3 ± 1.3 ms (n = 99 events), respectively (Fig. 3D). Kinetic analysis indicated that although the decay of the postsynaptic currents of the cell in culture for 6 days could be fitted by a single exponential function, the decay of the postsynaptic currents of the cell in culture for 15 days required two exponentials.
Figure 3. Electrophysiological properties of retinoic acid-treated mesenchymal stem cells. (A): Ongoing discharges recorded from a cell in culture for 4 days under current clamp condition. The resting membrane potential is at –50 mV. An accelerated trace is shown below. (B): Postsynaptic currents (PSCs) recorded at a holding potential of –60 mV from a cell in culture for 6 days. The trace below shows the decay of the PSC could be fitted by a single exponential. (C): PSCs recorded at a holding potential of –60 mV from a cell in culture for 15 days. The trace below in extended time scale shows the decay of the PSC could be fitted by two exponentials and has a time constant of 1 = 3.7 and 2 = 32.4 ms. The amplitudes were A1 = 3 pA and A2 = 24 pA. (D): Histogram of half-rise times and decay times of PSCs measured from cells in culture of 6 and 15 days.
Induction of SP in RA-Treated MSCs
This section describes studies to determine whether the neurons formed from MSCs could be induced to express a particular neurotransmitter. We selected IL-1 and the neurotransmitter gene PPT-I, because the regulation of PPT-I by inflammatory mediators such as IL-1 has been studied . Furthermore, the receptor for IL-1 has been reported in neurons . In addition, IL-1 can induce the production of SP, which is the major peptide derived from the PPT-I gene .
MSCs, treated with RA for 6 days, were stimulated with 10 ng/ml of IL-1 for 16 hours, and the cells were studied for SP production by immunofluorescence using anti-SP. The treated cells were also labeled for synaptovesicle 2, because it would be of interest to determine whether SP is stored in synaptic vesicle. The studies used MSCs exposed to RA for 6 days, because this time frame also showed functional synaptic activity (Fig. 3). RA-treated MSCs were labeled with anti-SP (FITC; Fig. 4, left column) and anti-synaptovesicle (PE; Fig. 4, middle panel). The two labels were overlaid to get insights on the location of SP. Because the anti-SP was diluted in 1% HSA and the immunofluorescence used indirect immunofluorescence, secondary antibody specificity was studied in parallel labeling with 1% HSA in lieu of anti-SP.
Figure 4. Immunohistochemistry for SP in MSC-derived neurons stimulated with IL-1. MSCs were treated with RA for 10 days and then stimulated with 25 ng/ml of IL-1 in the presence or absence of 4 μg/ml IL-RA. Control neurons contained vehicle for IL-1 (1% HSA). After 16 hours of stimulation, cells were labeled for SP by immunofluorescence. Shown are six experiments, each performed with MSCs from a different donor. Abbreviations: HSA, human serum albumin; IL, interleukin; IL-RA, IL-1 receptor antagonist; MSC, mesenchymal stem cell; RA, retinoic acid; SP, substance P.
Representations of six experiments, each performed with a different donor, are shown in Figure 4. The results show bright fluorescence intensity for SP in cells stimulated with IL-1 (Fig. 4, middle row). Fluorescence intensities were significantly reduced when the cells were costimulated with IL-1 RA (Fig. 4, bottom row). This indicates that the effects of IL-1 were specific. Cells labeled with 1% HSA showed minimal fluorescence, which was subsequently used as background fluorescence to compare other labeling studies (Fig. 4, top row). The results show that IL-1 can induce the production of SP in neurons generated from RA-treated MSCs.
Expression of ?-PPT-I in RA-Treated MSCs
Because dim fluorescence was observed for SP in studies with unstimulated MSCs treated with RA (not shown), we surmised that the gene from which SP is produced, PPT-I, might be expressed with low level of translation. We therefore asked whether the PPT-I gene was expressed in RA-treated MSCs by in situ hybridization with a cocktail of three oligomers (Table 1). Studies with neurons from three different BM donors are shown in Figure 5. Bright fluorescence was observed in the cell bodies of neurons (Fig. 5, right panels). Neurons labeled with sense oligonucleotides showed no fluorescence (not shown). The results indicate that the PPT-I gene is expressed in untreated MSCs differentiated with RA.
Figure 5. Expression of PPT-I in RA-treated MSCs. In situ hybridization for ?-PPT-I was performed with MSCs treated with RA for 10 days. Studies are shown for labeling with neurons generated from three different donors. Abbreviations: MSC, mesenchymal stem cell; PPT-I, preprotachykinin-I; RA, retinoic acid.
DISCUSSION
MSCs can be induced, via RA treatment, to become functional neurons that express neuron-specific phenotypic markers and exhibit synaptic properties similar to those of native neurons. The MSC-derived neurons express the tachykinin gene PPT-1 and can be induced to produce SP by IL-1 stimulation.
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