Stem cells may be a novel treatment modality for organ ischemia, possibly through beneficial paracrine mechanisms. Stem cells from older hosts have been shown to exhibit decreased function during stress. We therefore hypothesized that 1) neonatal bone marrow mesenchymal stem cells (nBMSCs) would produce different levels of IL-6, VEGF, and IGF-1 compared with adults (aBMSCs) when stimulated with TNF or LPS; 2) differences in cytokines would be due to distinct cellular characteristics, such as proliferation or pluripotent potential; and 3) differences in cytokines would be associated with differences in p38 MAPK and ERK signaling within nBMSCs. BMSCs were isolated from adult and neonatal mice. Cells were exposed to TNF or LPS with or without p38 or ERK inhibition. Growth factors were measured via ELISA, proliferation via daily cell counts, cell surface markers via flow cytometry, and pluripotent potential via alkaline phosphatase activity. nBMSCs produced lower levels of IL-6 and VEGF, but higher levels of IGF-1 under basal conditions, as well as after stimulation with TNF, but not LPS. Neonatal and adult BMSCs had similar pluripotent potentials and cell surface markers, but nBMSCs proliferated faster. Furthermore, p38 and ERK appeared to play a more substantial role in nBMSC cytokine and growth factor production. Neonatal stem cells may aid in decreasing the local inflammatory response during ischemia, and could possibly be expanded more rapidly than adult cells prior to therapeutic use.
- insulin-like growth factor-1
- vascular endothelial growth factor
- stem cell therapy
end-organ ischemia is a source of tremendous morbidity and mortality (8). Medical management of the more severe cases of ischemia is often inadequate, thereby warranting surgical resection of necrotic tissue or revascularization via arterial bypass. The subsequent reperfusion injury associated with the ischemic episode is also detrimental, and results in significant aberrations to the inflammatory cascade (21). In this regard, bone marrow mesenchymal stem cells (BMSCs) represent a novel treatment modality with increasing therapeutic potential (1, 12, 16, 22). Stem cells could be injected intravenously or directly into affected tissue prior to a planned ischemic episode, such as is seen in cardiac surgery. Conversely, cellular therapy may also be beneficial in the postischemic period, such as after myocardial infarction or bowel necrosis. The extensive proliferation and differentiation potential of BMSCs makes these cells optimal for seeding tissue-engineered grafts, which may allow for the replacement of damaged tissue or organs (25). In addition, stem cells have been shown to exhibit a variety of paracrine effects, including the release of protective growth factors and antiapoptotic signals (3, 27, 34).
Although BMSCs from hosts of varying ages are able to show multipotent potential, increasing age of stem cells and their hosts has been associated with decreased functional capacity under conditions of stress (2, 24). Specifically, increasing age has been associated with telomere shortening and dysfunction, mesenchymal progenitor cell dysfunction, a reduced capacity of bone marrow stromal cells to maintain functional hematopoeitic stem cells, and noted changes in cytokine production (15). It therefore seems logical that stem cells from younger hosts may be better adapted to stress during states of ischemia or endotoxemia and may provide increased growth factor production and better recovery after injury.
In an attempt to design the optimum stem cell for maximum therapeutic use, it becomes essential not only to elucidate the signaling mechanisms that BMSCs use to produce protective growth factors, but also to define the mechanisms used to enhance proliferation and to maintain viability. We therefore hypothesized that 1) neonatal BMSCs (nBMSCs) would produce different levels of IL-6, VEGF, and IGF-1 compared with adult BMSCs (aBMSCs) when stimulated with TNF or LPS; 2) differences in cytokine production would be associated with differences in distinct cellular characteristics, such as proliferation and pluripotent potential; and 3) differences in cytokine production would be associated with alterations in p38 MAPK and ERK signaling within the neonatal stem cell population.
MATERIALS AND METHODS
Normal adult (8–9 wk) and suckling (2½ wk) male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were acclimated in a quiet quarantine room. Adult mice had free access to tap water and standard mice chow. Neonatal mice remained with their birth mothers to breastfeed until the time of death. The animal protocol was reviewed and approved by the Indiana Animal Care and Use Committee of Indiana University. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 85-23, revised 1985).
Preparation of mouse bone marrow stromal cells.
A single-step stem cell purification method using adhesion to cell culture plastic was employed as previously described (23). Briefly, neonatal and adult mouse bone marrow stromal cells were collected from bilateral femurs and tibias after death by removing the epiphyses and flushing the shaft with complete media [Iscove's modified Dulbecco's medium (GIBCO Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (GIBCO Invitrogen)] using a syringe with a 26-gauge needle. Cells were disaggregated by vigorous pipetting several times and were passed through a 30-μm nylon mesh to remove remaining clumps of tissue. Cells were washed by adding complete media, centrifuging for 5 min at 300 rpm at 24°C, and removing supernatant. The cell pellet was then resuspended and cultured in 75 cm2 culture flasks with complete media at 37°C in 5% CO2 in air. BMSCs preferentially attached to the polystyrene surface; after 48 h, nonadherent cells in suspension were discarded. Fresh complete media were added and replaced every 3 or 4 days thereafter. When the cultures reached 90% of confluence, the stem cell culture was passaged; cells were recovered by the addition of a solution 0.25% trypsin-EDTA (GIBCO Invitrogen) and replated in flasks. Cells were utilized for experimentation between passages 3 and 9.
To ensure that experimental cells were of mesenchymal origin and to determine whether inherent differences existed in cellular surface markers, both adult and neonatal mesenchymal stem cells were assessed via flow cytometry. Cells were isolated from culture dishes using EDTA and were incubated with CD34, CD44, CD45, and CD117 primary antibodies (BD Biosciences, San Diego, CA) for 15 min. Following incubation, the reaction was stopped by adding cold PBS with 1% FBS. Cells were then centrifuged at 400 g, and the PBS solution was decanted. Cells were resuspended in fresh PBS and assayed via flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences). Experiments were performed at least three times to ensure accurate results.
BMSCs were plated in 12-well plates at a concentration of 1 × 105 cells/well/ml. Adult and neonatal cells were divided into three experimental groups (triplicate wells per group) and stressed with 1) no stimulus, 2) TNF (50 ng/ml), or 3) LPS (100 ng/ml) to cause cellular distress. Concentrations of stimulants were determined from previous studies examining adult stem cell mechanisms (30). Cells were subsequently exposed to p38 or ERK inhibition [SB203580, SB202190, ERK Inhibitor I, ERK Inhibitor II, (10 μM) (Calbiochem, San Diego, CA)] with or without the addition of the above stimulants. After 24 h of incubation, supernatants were harvested for molecular assay. Experiments were repeated several times for verification of results (total n = 6–21/group).
IL-6, VEGF, and IGF-1 in supernatants were determined by ELISA using a commercially available ELISA set (R&D Systems, Minneapolis, MN; BD Biosciences). ELISA was performed according to the manufacturer's instructions. All samples and standards were measured in duplicate.
Cellular proliferation was assessed by constructing cellular growth curves. Briefly, 1,000 cells were plated into 12-well plates with media. Each day, cells were recovered from three wells of the plate by the addition of a solution 0.25% trypsin-EDTA (GIBCO Invitrogen). Trypan blue was then added to a 20-μl aliquot of cell solution, and viable cells were counted with the aid of a hemocytometer. Each well was counted in duplicate. Three curves were constructed for each cell line (passages 4–7), and the average growth was plotted.
The potential for pluripotency within neonatal and adult mesenchymal stem cells was assessed by enzyme-labeled fluorescence alkaline-phosphatase staining (American Type Culture Collection, Manassas, VA). Highly undifferentiated stem cells exhibit strong alkaline phosphatase activity. Staining was performed according to manufacturer's instructions and viewed using a standard Hoescht/DAPI long-pass filter set. Light microscopy was performed to ensure the presence of cells on the slide and to characterize morphology. Experiments were performed three times to ensure accurate results.
Presentation of data and statistical analysis.
All reported values are expressed as means ± SE. Data were compared using Student's t-test. A probability value of less than 0.05 was considered statistically significant.
Neonatal and adult stem cells express mesenchymal stem cell surface markers.
Stem cells that were harvested from adult and neonatal mice maintained similar mesenchymal stem cell surface markers. Both cell lines were negative for the hematopoietic stem cell markers CD34, CD45, and CD117, but they were positive for the mesenchymal stem cell marker CD44 (Table 1).
Neonatal stem cells produce less IL-6 and VEGF, but more IGF-1.
TNF and LPS stimulated both neonatal and adult BMSCs to produce IL-6, VEGF, and IGF-1. After stimulation with TNF though, neonatal stem cells produced significantly less IL-6 and VEGF, and significantly more IGF-1 compared with adults. There was no significant difference in LPS-stimulated levels of IL-6, VEGF, or IGF-1 between neonatal and adult stem cells. Basal levels of IL-6 and VEGF were significantly lower in neonates compared with adults, while basal levels of IGF-1 were higher in neonates (Fig. 1).
Morphology, pluripotent potential, and proliferation.
Neonatal and adult stem cells maintained similar cell morphology in normal culture conditions. Unlike embryonic stem cells, which were morphologically smaller and grew in distinct clumps, both adult and neonatal mesenchymal stem cells adhered to plastic and grew in a monolayer until confluent. In addition, nBMSCs and aBMSCs did not express alkaline phosphatase activity, thereby indicating a lower degree of pluripotent potential compared with the more totipotent embryonic stem cell (Fig. 2).
Neonatal BMSCs were noted to proliferate faster than their adult counterparts over the course of the 12 days observed. Neonates proliferated faster at all time points, with statistically higher rates of proliferation being noted at days 5 (neonate: 81,175 ± 325 cells/ml vs. adult: 17,889 ± 12,094 cells/ml) and 8 (neonate: 102,833 ± 25,563 cells/ml vs. adult: 26,394 ± 5,743 cells/ml) (Fig. 3).
p38 and ERK regulate stem cell cytokine and growth factor production.
p38 was noted to play a critical role in the production of stimulated IL-6 in adults, as well as IL-6 and VEGF in neonates. Specifically, stimulated IL-6 production in adults, and IL-6 and VEGF in neonates, were significantly lower with the addition of either p38 inhibitor (SB203580 or SB202190). VEGF levels in adults, as well as IGF-1 production in both adults and neonates, were slightly decreased with p38 inhibition, but these values did not reach statistical significance (Fig. 4).
Adult levels of TNF and LPS stimulated IL-6 decreased with the addition of ERK inhibitor II, which inhibits both ERK 1 and ERK 2. Furthermore, neonatal IL-6, VEGF, and IGF-1 levels all decreased significantly with the addition of ERK inhibitor II. ERK inhibitor I, which only inhibits ERK 2, had no effect on growth factor production on either cell line, with the exception that it increased neonatal IL-6 production after stimulation with LPS. (Fig. 5).
Mesenchymal stem cells are a potent source of protective growth factors and may be useful in the treatment of a variety of planned or unplanned ischemic episodes, including prior to coronary artery bypass grafting or following end-organ infarction. Herein, we discovered that 1) mesenchymal stem cells harvested from neonatal hosts produce significantly lower levels of IL-6 and VEGF and higher levels of IGF-1 under basal conditions and after stimulation with TNF; 2) nBMSCs proliferate more rapidly than aBMSCs yet maintain similar cell surface markers and pluripotent potential; and 3) p38 and ERK are more prominent in regulating production of cytokines and growth factors in neonatal BMSCs compared with adults.
Many cytokines are elevated during conditions of ischemia or endotoxemia, and stimulating stem cells with TNF or LPS is an adequate means to simulate these harsh conditions in cell culture (3, 7, 38). Cellular expression of IL-6 is elevated in various pathologies, including sepsis, ischemia, and trauma (13, 18–20, 26), and multiple studies have suggested that lower levels of IL-6 are associated with better outcomes and improved recovery after an ischemic injury (3, 9, 31, 32, 35). IL-6 production was increased in both neonates and adults after stimulation with TNF or LPS and likely represents activation of the acute-phase response. Basal and TNF-induced IL-6 production from neonates, however, was lower than that of the adults, while LPS stimulated IL-6 levels were similar between adults and neonates. Previous studies have noted elevated cytokines in aging stem cells, and therefore, lower expression of IL-6 may suggest either a decreased inflammatory response within nBMSCs, or conversely, a heightened response in adults.
VEGF production was noted to be decreased in basal and TNF-stimulated neonates compared with adults. VEGF has been shown to increase angiogenesis and to promote stem cell survival (10, 11, 28, 33). Previous in vivo studies using stem cell therapy have demonstrated an association between elevated postischemic levels of VEGF and improved function (4, 6). VEGF was therefore expected to be higher in the younger neonatal stem cell line. The decreased levels of stimulated VEGF exhibited by neonates may actually suggest that these cells are not superior to adult stem cells in providing protection to ischemic tissues.
Neonatal levels of IGF-1 were also shown to be elevated compared with adults under normal and TNF-stimulated culture conditions. IGF has been implicated as an important mediator in stem cell survival and self-renewal (29). In this study, neonatal stem cells were observed to proliferate faster than adults under basal conditions associated with elevated IGF-1 production. These data corroborate previous studies on human mesenchymal stem cells, which showed that BMSCs harvested from younger hosts proliferate faster than adults (17). However, during noxious stimulation with TNF or LPS, IGF-1 levels were noted to decrease in both cell lines. This may indicate that cells shifted from a phase of self-renewal to a phase of self-conservation following noxious stimulation.
nBMSCs and aBMSCs shared a few similar characteristics, including alkaline phosphatase activity, cell surface markers, and cellular morphology. Alkaline phosphatase activity, which is normally seen in highly undifferentiated cells, such as embryonic stem cells, was noted to be absent in both neonatal and adult BMSCs. We surmised that this activity would be present in nBMSCs because they were derived from younger hosts. Unexpectedly though, alkaline phosphatase activity was not present in either cell line, thereby indicating that both cell lines have a lower pluripotent potential compared with embryonic stem cells. Measured cell surface markers confirmed that both lines were of mesenchymal stem cell origin. CD34, CD45, and CD117, which are normally negative on mesenchymal stem cells, as well as CD44, which is normally positive on mesenchymal stem cells, were also similar between neonates and adults. These data would indicate that these stem cell markers do not change with increasing age of the host (14, 36, 37).
Our previous studies suggested that p38 MAPK and ERK activation was increased in aBMSCs following noxious stimulation (5, 30). In this study, both kinases were observed to regulate cytokine production within each cell line but seemed to play a more substantial role within nBMSCs. p38 appeared essential for IL-6 production in aBMSCs, as well as IL-6 and VEGF production in neonates. However, p38 did not appear to be essential for the production of IGF-1 in either cell line. It is unclear though, as to why p38 was not involved in aBMSC VEGF production, as previous studies from our lab have suggested that VEGF production was dependent on the p38 arm of the MAPK pathway (30). The cells used for those previous studies, however, were of human origin, and it is possible that different MAPK signaling cascades exist between human and mouse BMSCs.
ERK also appeared to play a role in regulating adult and neonatal IL-6 production, as well as neonatal IL-6, VEGF, and IGF-1 production. Specifically, ERK inhibitor II, which inhibits both ERK 1 and ERK 2, was noted to significantly decrease the aforementioned growth factors, while inhibition of ERK 2 alone did not decrease growth factor production. This observation would suggest that ERK 1 is the major isoform of ERK that plays a role in cytokine and growth factor production following noxious stimulation. In this regard, overexpression of ERK 1 may work to increase beneficial growth factor production within stem cells, thereby providing even greater protection from ischemia during therapeutic use.
In conclusion, BMSCs harvested from younger hosts, such as neonates, are a potent source of protective cytokines and growth factors that could potentially be harnessed for the treatment of a variety of ischemic conditions. Certain growth factors, such as IL-6 and VEGF, were decreased in nBMSCs during basal conditions and after noxious stimulation, while others, such as IGF-1, were increased. Further studies are needed to differentiate the cytokines and growth factors that are most important in promoting increased survival and postinjury recovery. Understanding the mechanisms that BMSCs use to produce these protective growth factors will allow for the engineering of “super stem cells” designed for optimal growth factor production and maximal ex vivo priming prior to therapeutic use.
This work was supported in part by National Institutes of Health R01 GM070628, R01 HL-085595, K99/R00 HL-0876077-01, NRSA F32 HL085982, an American Heart Association (AHA) Grant in aid, and AHA Postdoctoral Fellowship 0526008Z.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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