The secretome of stem cells isolated from the adipose tissue and Wharton jelly acts differently on central nervous system derived cell populations
© Ribeiro et al.; licensee BioMed Central Ltd. 2012
Received: 17 February 2012
Accepted: 2 May 2012
Published: 2 May 2012
It is hypothesized that administration of stromal/stem cells isolated from the adipose tissue (ASCs) and umbilical cord (HUCPVCs) can ameliorate the injured central nervous system (CNS). It is still not clear, however, whether they have similar or opposite effects on primary cultures of neuronal populations. The objective of the present work was to determine if ASCs and HUCPVCs preferentially act, or not, on specific cell populations within the CNS.
Primary cultures of hippocampal neurons were exposed to ASCs and HUCPVCs conditioned media (CM) (obtained 24, 48, 72 and 96 hours after three days of culture) for one week.
Cell viability experiments (MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H tetrazolium) test) revealed that CM obtained from both cell populations at all time points did not cause any deleterious effects on neuronal cells. In fact, it was determined that whenever the ASCs CM were supplemented with basic fibroblast growth factor (bFGF) and B27, there was a significant increase in the metabolic viability and neuronal cell density of the cultures. On the other hand, in the absence of CM supplementation, it was the HUCPVCs secretome that had the highest impact on the metabolic viability and cell density. In an attempt to unveil which factors could be involved in the observed effects, a screening for the presence of bFGF, nerve growth factor (NGF), stem cell factor (SCF), hepatocyte growth factors (HGF) and vascular endothelial growth factor (VEGF) in the CM was performed. Results revealed the presence of all these factors in ASCs CM, except bFGF; in contrast, in HUCPVCs CM it was only possible to detect robust NGF expression.
Overall, the results confirm important differences on the secretome of ASCs and HUCPVCs, which lead to distinct effects on the metabolic viability and neuronal cell densities in primary cultures of hippocampal neurons; however, the factor(s) that promote the stronger effect of the HUCPVCs CM in neuronal survival is(are) still to be identified.
Currently there are no effective treatments for major central nervous system (CNS) injuries/disorders . In the last decade, stem/progenitor cells isolated from the adipose tissue (ASCs) and the Wharton jelly of the umbilical cord have been proposed for possible transplantation as a therapy for CNS injuries [2–4]. Presently, it is commonly accepted that their potency is related mainly to their secretome, that is, to the production of molecules with a neuroregulatory character that support neuronal/glial cell survival and create an environment conducive to regeneration by endogenous cells [2, 3].
Salgado et al.  demonstrated that the conditioned media (CM) of a population of mesenchymal progenitors isolated from the Wharton jelly, located in the perivascular region of the umbilical cord (human umbilical cord perivascular cells - HUCPVCs), were able to increase cell viability, survival and proliferation in primary cultures of hippocampal neurons and glial cells. Koh et al.  also revealed that the expression of granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), glial derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF) could be correlated with the neuroprotector effect revealed by stem cells isolated from the bulk of the Wharton jelly (WJ-MSCs), when transplanted to animal models of brain ischemia. Similar findings were also reported by Ding et al.  in an animal model of ischemic stroke. In this case the transplantation of human WJ-MSCs was not only able to promote functional recovery of behavioral deficits, but also the reduction of the lesion size, a higher extent of vascularization in ischemic areas and finally a higher expression of Stem Cell Derived Factor 1 (SDF-1), BDNF and GDNF in ischemic tissues. Identical outcomes were also observed in other animal models of injury within the CNS. For instance, Yang and colleagues  reported the improvement of spinal cord injured rats upon transplantation of undifferentiated WJ-MSCs and related these results with the expression of human neutrophil-activating protein-2 (NAP-2), neurotrophin-3 (NT-3), basic fibroblast growth factor (bFGF), glucocorticoid induced tumor necrosis factor receptor (GITR) and vascular endothelial growth factor receptor 3 (VEGFR-3). Finally, Weiss et al.  also revealed that WJ-MSCs could induce an overall improvement in the condition of an animal model of Parkinson's Disease (PD) through an increase of expression of GDNF at the site of injury.
Similar to what has been reported for stem cells isolated from the WJ's UC, growth factors such as VEGF, hepatocyte growth factor (HGF), bFGF, insulin like growth factor (IGF-1) and others have also been found in the ASCs secretome [10–12].
The ASCs application in models of injury, neurodegeneration and neurotoxicity is also well described. For instance Lee et al.  showed that ASCs transplantation into a mice model of Huntington Disease (HD) slowed down the disease progression by modulating the host pathogenesis. Lu and colleagues  also revealed that ASCs secretome exerted neuroprotection on glutamate mediated excitotoxicity in a PC12 cell line model. Moreover, this was partially related to the presence of different levels of BDNF, VEGF and HGF . Another study using the PC12 cell line also reported interesting results . In this particular work it was observed that ASCs conditioned media was able to induce cell neuritogenesis, and that this effect was partially mediated through a NGF related mechanism. Finally, ASCs application to a rat model of brain hypoxic-ischemic injury was also reported by Wei et al. . Their objective was to study the role of concentrated conditioned media from cultured rat ASCs (ASC-CM) on the protection/recovery of the model. Both behavioral analysis and post-mortem evaluation of brain damage revealed that the conditioned media had a neuroprotective character. In this case IGF-1 and BDNF were indicated as the main mediators of the observed effects.
As it was shown, the effects of these cells on different CNS cell populations are relatively well described. However, a direct comparison of the effects of their secretome on CNS cells has not been performed. Therefore, the objective of the present work was to determine and compare the effects of the secretome of human ASCs and HUCPVCs on primary cultures of post-natal rat hippocampal neurons. For this purpose the latter were incubated with either conditioned media (CM) from ASCs or HUCPVCs. Results revealed that both ASCs and HUCPVCs secretome are able, in distinct forms, to increase cell viability and cell densities in the tested culture system, a fact that can be considered as an indicator of significant differences in their secretome composition.
Adipose tissue derived stem cells
ASCs were isolated according to a protocol previously described by Dubois et al. . All protocols were reviewed and approved by the Pennington Biomedical Research Center Institutional Research Boards (IRB) prior to the study. Liposuction aspirates from subcutaneous adipose tissue sites (abdomen, flank, thighs) were obtained from female subjects undergoing elective plastic surgical procedures. All donors gave their written informed consent. Tissues were then digested in a 0.1% collagenase type I solution (Worthington Biochemical Corporation, Lakewood, NJ, USA) pre-warmed to 37°C for 60 minutes, after which they were centrifuged for five minutes at 300 g to 500 g at room temperature. The supernatant, containing mature adipocytes, was aspirated. The pellet was identified as the stromal vascular fraction (SVF). The SVF was resuspended and plated immediately in T225 flasks in Stromal Medium [DMEM/F 12 Ham's, 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 U penicillin/100 μg streptomycin/0.25 μg Fungizone ] at a density of 0.156 ml of tissue digest/cm2 of surface area for expansion and culture. After reaching confluence, cells were passaged and kept in stromal medium.
Human umbilical cord perivascular cells
HUCPVCs were isolated from the UCs of local consenting full-term caesarean section patients. Ethical approval had been previously obtained from Hospital de S. Marcos, Braga, Portugal. Parents gave their written informed consent prior to the umbilical cord collection. Cells were isolated according to the procedure originally described by Sarugaser et al. . Pieces of cord, 4 to 5 cm long, were dissected by first removing the epithelium of the UC section along its length to expose the underlying WJ. Each vessel, with its surrounding WJ matrix, was then pulled away and incubated in a 0.5 to 0.75 mg/ml collagenase (Sigma, St. Louis, MO, USA) with PBS (Gibco, Grand Island, NY, USA) solution for 18 hours. Supernatant was then diluted with PBS to reduce the viscosity of the suspension and centrifuged. Cells were further resuspended in culture media [α-MEM (Gibco) supplemented with 10% FBS (Gibco) and 1% antibiotic/antimycotic (Sigma)], plated in T75 flasks at a density of 4,000 cells/cm2. The culture medium was changed every two to three days. Upon confluence cells were trypsinized and passaged to new T75 flasks.
Hippocampal neuronal cultures were prepared from P4 Wistar rats . Briefly, upon dissection, hippocampi were submitted to a trypsin based enzymatic digestion followed by mechanical dissociation. Isolated cells were then plated on Poly-D-Lysine (Sigma) coated coverslips at a density of 4,000 cells/cm2. Cultures were then incubated with CM and respective controls, as described in 2.2.
Conditioned medium collection and experiments
CM was collected from P5 ASCs and HUCPVCs. For this purpose cells were plated out at a density of 4,000 cells/cm2 and allowed to grow for three days. On day three, culture medium was renewed and CM were collected 24, 48, 72 and 96 hours thereafter and frozen. For CM collection Neurobasal-A medium supplemented with kanamycin (Gibco, 0.1 to mg/ml) was the chosen medium. Upon isolation, hippocampal neurons were plated out at the densities referred above and incubated from T0 with the previously collected and filtered CM (n = 3/CM time point) for seven days, after which cell viability and differentiation were assessed (see below). Two experimental setups were outlined: 1) B27 (Gibco) supplement and bFGF (Gibco, 10 ng/ml) were added to the CM prior to neuron incubation (control: standard Neurobasal-A media supplemented with the same concentrations of bFGF, B27 and kanamycin) and 2) CM were used as taken from the cultures flasks, without the addition of the supplements referred in 1).
Cell viability assessment
Cell viability was assessed by the MTS test. The MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H tetrazolium) (Promega, Madison, WI, USA) test is an assay in which the substrate, MTS, is bioreduced into a brown formazan product by nicotinamide adenine dinucleotide phosphate (NADPH) or NADP produced by mitochondrial enzymes, which are active in living cells. Cell culture coverslips (n = 3) were placed in culture medium containing MTS in a 5:1 ratio and incubated in a humidified atmosphere at 37ºC and 5% CO2. After three hours of incubation, 100 μl of solution from each sample was transferred to 96 well plates and the optical density was determined at 490 nm. Results are shown as a ratio between CM and control incubated cultures.
Cells were fixed in 4% paraformaldehyde for 30 minutes, permeabilized by incubation with 0.3% Triton X-100 in PBS for five minutes at room temperature (for neurons and astrocytes), and washed three times in PBS. Cells were then blocked with 10% FBS/PBS (60 minutes), followed by a 60-minute incubation with a mouse anti-rat microtubule associated protein 2 (MAP-2) (Sigma) antibody in order to detect mature hippocampal neurons. Cells were then washed in PBS and incubated with Alexa Fluor 594 goat anti-mouse immunoglobulin G (IgG). Primary antibody was omitted to produce negative controls. Samples were observed under an Olympus BX-61 Fluorescence Microscope (Olympus, Germany).
Cell counts were performed by using Cell-P software (Olympus, Germany). For this purpose three cover slips per condition and three representative fields were chosen and analyzed. Results are shown as a ratio between the percentage of MAP-2 positive cells found in CM and control incubated cultures, respectively.
Analysis of the conditioned media of ASCs and HUCPVCs
CM were assayed for cytokines using a Bio-plex human 5-plex panel immunoassay kit (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instructions. The 5-plex panel consisted of the following analytes: bFGF, VEGF, NGF, SCF and HGF. A standard range of 0.2 to 3,200 pg/mL was used. CM samples were collected as previously described, centrifuged and frozen. Samples and controls were run in triplicate, standards and blanks in duplicate. Results were normalized to the total protein present in the respective CM.
Statistical evaluation was performed using one way (hippocampal neurons experiments) analysis of variance (ANOVA) tests to assess the statistical differences. Statistical significance was defined as P < 0.05.
Results and discussion
The present study set out to determine to what extent the secretome of two populations of human stromal/stem cells, ASCs and HUCPVCs, influence the viability and cellular densities of rat post-natal hippocampal neurons.
Quantification of bFGF, VEGF, NGF, SCF and HGF in ASCs and HUCPVCs CM (n = 3 + SD, results shown in pg/g of protein).
ASCs CM24 h
50,174 ± 52,735
22,152 ± 1,768
1,028 ± 891
ASCS CM48 h
49,543 ± 39,746
1,872 ± 1,305
1,740 ± 2,699
1,626 ± 1,098
ASCs CM72 h
26,441 ± 16,484
1,800 ± 1,637
2,949 ± 2,546
4,752 ± 4,337
ASCs CM 96 h
27,984 ± 19,191
1,488 ± 835
2,311 ± 1,232
4,269 ± 5,036
HUCPVCs CM24 h
1,942 ± 1,325
HUCPVCs CM48 h
2,630 ± 1,520
HUCPVCs CM72 h
730 ± 537
2,139 ± 251
HUCPVCs CM96 h
1,311 ± 1,153
The present work demonstrates that ASCs and HUCPVCs release trophic/neuroregulatory factors that improve the metabolic viability of hippocampal cultures. Importantly, it was possible to observe that their secretome acts differently on the cell viability and densities of post-natal cultures of hippocampal neurons. ASCs secretome effects are dependent on the interaction with added exogenous factors such as bFGF, while, on the other hand, HUCPVCs secretome is able to promote neuronal survival/differentiation in the absence of exogenous supplements.
adipose tissue derived stem cells
brain derived neurotrophic factor
basic fibroblast growth factor
central nervous system
fetal bovine serum
granulocyte colony-stimulating factor
glial derived neurotrophic factor
hepatocyte growth factor
human umbilical cord perivascular cells
insulin like growth factor 1
microtubule associated protein 2
neutrophil-activating protein 2
nerve growth factor
stem cell factor
stem cell derived factor 1
stromal vascular fraction
vascular endothelial growth factor
vascular endothelial growth factor receptor 3
Wharton jelly mesenchymal stem cells.
Foundation Calouste de Gulbenkian for funds under the scope of the The Gulbenkian Programme to Support Research in the Life Sciences and Ciência 2007 Program from the Portuguese Foundation for Science and Technology (AJS); Pennington Biomedical Research Foundation (JMG); James Wade MD, his office staff, and patients for donation of the lipoaspirates. This work was performed following the terms of the cooperation agreement signed between the 3B's Research Group of the University of Minho and the Hospital de São Marcos in Braga and approved by its ethical committee.
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