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Brain-Derived Neurotrophic Factor (BDNF) Produced by Human Umbilical Tissue-derived Cells (hUTC) Is Required for its Effect on Hippocampal Dendritic Differentiation
Alder, Janet; Kramer, Brian C.; Hoskin, Casey; et.al. https://scholarship.libraries.rutgers.edu/discovery/delivery/01RUT_INST:ResearchRepository/12643413340004646?l#13643549350004646
Alder, J., Kramer, B. C., Hoskin, C., & Thakker-Varia, S. (2012). Brain-Derived Neurotrophic Factor (BDNF) Produced by Human Umbilical Tissue-derived Cells (hUTC) Is Required for its Effect on Hippocampal Dendritic Differentiation. In Developmental Neurobiology (Vol. 72, pp. 755–765). Rutgers University. https://doi.org/10.7282/T30P1206
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Brain -Derived Neurotrophic Factor (BDNF) Produced by Human Umbilical Tissue-derived Cells (hUTC) Is Required for its Effect on Hippocampal Dendritic Differentiation
For Peer Review Journal: Developmental Neurobiology
Manuscript ID: Neuro-00133-2011.R1
Wiley - Manuscript type: Research Article
Date Submitted by the Author: n/a
Complete List of Authors: Alder, Janet; UMDNJ - RWJ, Neuroscience and Cell Biolgoy Kramer, Brian; Advanced Technologies and Regenerative Medicine, LLC Hoskin, Casey; UMDNJ - RWJ, Neuroscience and Cell Biolgoy Thakker-Varia, Smita; UMDNJ - RWJ, Neuroscience and Cell Biolgoy
Cell therapy, neurotrophin, dendritic differentiation, synaptogenesis, Key Words: growth factor
John Wiley & Sons, Inc. Page 1 of 37 Developmental Neurobiology
1 2 3 Brain-Derived Neurotrophic Factor (BDNF) Produced by Human Umbilical 4 5 6 Tissue-derived Cells (hUTC) Is Required for its Effect on Hippocampal 7 8 Dendritic Differentiation 9 10 11 12 13 Running title: hUTC secrete BDNF to induce dendrites 14 15 16 17 1 2 1 1 18 Janet Alder, BrianFor C. Kramer, PeerCasey Hoskin, Review Smita Thakker Varia 19 20 21 22 1 Department of Neuroscience and Cell Biology, University of Medicine and 23 24 25 Dentistry of New Jersey Robert Wood Johnson Medical School, Piscataway, 26 27 New Jersey 08854 28 29 30 31 2 32 Advanced Technologies and Regenerative Medicine, LLC, an affiliate of 33 34 Johnson & Johnson, PO Box 151, Somerville, New Jersey 08876 35 36 37 38 39 Correspondence to: Smita Thakker Varia, PhD, Department of Neuroscience and 40 41 Cell Biology, University of Medicine and Dentistry of New Jersey Robert Wood 42 43 44 Johnson Medical School, 675 Hoes Lane West, RWJSPH 357A, Piscataway, 45 46 New Jersey 08854, Phone: (732) 235 5393, Fax: (732) 235 4990, Email: 47 48 [email protected] 49 50 51 52 53 Acknowledgements: We would like to thank Jennifer Jernstedt Ayer (UMDNJ) 54 55 and Chin Feng Yi (a former member of ATRM) for technical assistance. 56 57 58 59 60 1 John Wiley & Sons, Inc. Developmental Neurobiology Page 2 of 37
1 2 3 ABSTRACT 4 5 6 The potential for non embryonic cells to promote differentiation of neuronal cells 7 8 has therapeutic implications for regeneration of neurons damaged by stroke or 9 10 11 injury and avoids many ethical and safety concerns. We assessed the capacity 12 13 of human umbilical tissue derived cells (hUTC) and human mesenchymal stromal 14 15 cells (hMSC) to enhance differentiation of rodent hippocampal neurons. Co 16 17 18 culture of hippocampalFor cells Peer with hUTC orReview hMSC in transwell inserts for 3 days 19 20 resulted in increase of several dendritic parameters including the number and 21 22 length of primary dendrites. The effect of hUTC or hMSC on dendritic 23 24 25 maturation was only apparent on neurons grown for two weeks in vitro prior to 26 27 co culture. Changes in dendritic morphology in the presence of hUTC were also 28 29 accompanied by increased expression of the presynaptic marker synaptotagmin 30 31 32 and the postsynaptic marker postsynaptic density protein 95kD (PSD95) 33 34 suggesting that there may also be an increase in the number of synapses formed 35 36 37 in the presence of hUTC. The effect of hUTC and hMSC on hippocampal cells in 38 39 co culture was comparable to those induced by treatment with recombinant 40 41 human BDNF (rhBDNF) implying that a similar factor may be released from 42 43 44 hUTC or hMSC. Analysis of hUTC conditioned medium by ELISA demonstrated 45 46 that BDNF was indeed secreted. An antibody that blocks the actions of BDNF 47 48 partially inhibited the actions of hUTC on dendritic morphology suggesting that 49 50 51 BDNF is at least one of the factors secreted from the cells to promote dendritic 52 53 maturation. These results indicate that hUTC secrete biologically active BDNF 54 55 which can affect dendritic morphology. 56 57 58 59 60 2 John Wiley & Sons, Inc. Page 3 of 37 Developmental Neurobiology
1 2 3 4 5 6 KEYWORDS: Cell therapy, neurotrophin, dendritic differentiation, 7 8 synaptogenesis, growth factor 9 10 11 12 13 INTRODUCTION 14 15 The ability of hippocampal cells to respond to environmental signals and change 16 17 18 their dendritic morphologyFor Peermay allow the Review neurons to recover following an insult 19 20 such as stroke or injury. The morphological events of hippocampal neuronal 21 22 maturation in culture are well characterized and can be used to study the effects 23 24 25 of exogenous factors on neuronal differentiation and regeneration. Primary 26 27 dendrites protrude from the cell body starting from 1 day in vitro (div) until 10 div. 28 29 Secondary dendritic branches then extend from the primary branches between 30 31 32 approximately 6 12 div forming a network of neurites. Finally, spine formation 33 34 and maturation occurs from 12 21 div resulting in synaptic contacts (Dotti et al., 35 36 37 1988; Craig and Banker, 1994). Dendritic branching can be elaborated during 38 39 development as well as modified in the mature brain (Horch, 2004). There is an 40 41 overwhelming amount of evidence that synapses are plastic and undergo short 42 43 44 and long term modifications (Yuste and Bonhoeffer, 2001; Segal, 2005) at both 45 46 the pre and post synaptic sites leading to neuronal morphological changes. 47 48 Extrinsic factors implicated in alterations in dendritic branching include 49 50 51 membrane bound as well as soluble compounds such as neurotrophins 52 53 (Landgraf and Evers, 2005). 54 55 56 57 58 59 60 3 John Wiley & Sons, Inc. Developmental Neurobiology Page 4 of 37
1 2 3 BDNF is highly expressed in the hippocampus in an activity dependent 4 5 6 manner (Isackson et al., 1991; Lindholm et al., 1994; Berninger et al., 1995; West 7 8 et al., 2001) and has been implicated in the processes of dendritic branching and 9 10 11 spine development, functional maturation of synapses as well as synaptic 12 13 plasticity of adult hippocampal neurons (Cohen Cory, 2002). BDNF affects the 14 15 dendritic branching of several cell types including excitatory pyramidal neurons 16 17 18 (McAllister et al.,For 1995; LabellePeer and Leclerc, Review 2000; Alonso et al., 2004) and 19 20 increases the number of primary dendrites in pyramidal but not in nonpyramidal 21 22 cells neurons (McAllister et al., 1995; McAllister et al., 1996; Horch et al., 1999). 23 24 25 The requirement for the BDNF receptor trkB, in dendritic morphogenesis has 26 27 been demonstrated (Martinez et al., 1998; Gascon et al., 2005; Zagrebelsky et 28 29 al., 2005) and several signaling molecules downstream of trkB have been 30 31 32 implicated in the regulation of dendritic size and complexity by BDNF (Ng et al., 33 34 2006). In addition, BDNF promotes the formation of hippocampal synapses 35 36 37 (Vicario Abejon et al., 1998; Marty et al., 2000; Seil and Drake Baumann, 2000; 38 39 Collin et al., 2001; Elmariah et al., 2004) and increases presynaptic (Mizuno et 40 41 al., 1994; Altar, 1999; Tartaglia et al., 2001; Aguado et al., 2003) as well as 42 43 44 postsynaptic (Yamada et al., 2002; Elmariah et al., 2004) gene expression and 45 46 differentiation. Once a synapse has formed, BDNF has well defined effects on 47 48 synaptic activity in the hippocampus (Lessmann et al., 1994; Levine et al., 1995; 49 50 51 Lu and Figurov, 1997; Takei et al., 1997; Black, 1999; Sherwood and Lo, 1999; 52 53 Schinder et al., 2000). These relatively fast changes in synaptic efficacy may be 54 55 56 57 58 59 60 4 John Wiley & Sons, Inc. Page 5 of 37 Developmental Neurobiology
1 2 3 translated into structural alterations when the synapses are exposed to BDNF for 4 5 6 a longer period of time (Martinez et al., 1998; Pozzo Miller et al., 1999). 7 8 9 10 11 Following stroke or other CNS injury there is extensive neuronal death and 12 13 degeneration. Effective treatment for stroke is still elusive although attention is 14 15 increasingly focused on cell based therapies. Transplantation of stem cells 16 17 18 following experimentallyFor inducedPeer stroke Review promotes cell survival, repair of 19 20 degenerating neurons as well as functional recovery (Grabowski et al., 1993; 21 22 Chen et al., 2001; Li et al., 2001; Savitz et al., 2002; Joyce et al., 2010; Barkho 23 24 25 and Zhao, 2011; Luo, 2011). Ethical and safety issues surrounding the use of 26 27 embryonic stem cells has stimulated the investigation of alternative sources of 28 29 cells for the treatment of stroke. The use of cells derived from human umbilical 30 31 32 cord tissue as well as human bone marrow derived mesenchymal stromal cells 33 34 represent promising tools for new clinical concepts in supporting cellular therapy. 35 36 37 Human umbilical tissue derived cells (hUTC) can also be obtained by rather less 38 39 invasive methods and avoid many ethical and safety concerns. Previous studies 40 41 suggest that administration of hUTC (Zhang et al., 2011) or hMSC (Koh et al., 42 43 44 2008; Secco et al., 2009) promotes recovery following stroke including 45 46 neurogenesis, increased synaptic and vascular densities and restoration of 47 48 neurological function. The current study examines the effects of hUTC or hMSC 49 50 51 on hippocampal neuronal differentiation in vitro suggesting paracrine actions of 52 53 these cells. We demonstrate that hUTC as well as hMSC promote dendritic 54 55 differentiation in more mature hippocampal neurons as compared to control and 56 57 58 59 60 5 John Wiley & Sons, Inc. Developmental Neurobiology Page 6 of 37
1 2 3 that hUTC enhance synaptic marker expression similar to the actions of BDNF. 4 5 6 We further show that hUTC secrete BDNF and that BDNF contributes to the 7 8 effects of hUTC on hippocampal cell differentiation. 9 10 11 12 13 METHODS 14 15 16 17 18 Hippocampal NeuronalFor Culture Peer Review 19 20 Cultures were obtained from E18 rat hippocampi (Thakker Varia et al., 2001). 21 22 Embryonic day 18 rat fetuses were removed from CO sacrificed rats (Hilltop 23 2 24 25 Laboratories) by cesarean section in accordance with institutional guidelines for 26 27 care and use of animals and transferred to sterile PBS containing Petri dishes. 28 29 The hippocampi were dissected out from surrounding brain tissue, and the 30 31 32 meninges were completely removed. Pooled tissue from each litter was 33 34 mechanically dissociated in 0.5 ml MEM containing 7.5% fetal bovine serum and 35 36 37 1.5 ml serum free medium consisting of Neurobasal medium, B27 supplement, 38 39 penn/strep and L glutamine (Invitrogen). Cells were diluted in the serum free 40 41 medium and plated on poly D lysine coated dishes at 250,0000 cells/well in a 6 42 43 44 well dish in a final volume of 2.6 ml, and maintained at 37 ºC in a 95% air/5% 45 46 CO 2 humidified incubator. 47 48 49 50 51 Preparation of hUTC or hMSC 52 53 Human umbilical tissue derived cells (hUTC) were isolated and banked as 54 55 56 previously described (Zhang et al., 2011). Human bone marrow derived 57 58 59 60 6 John Wiley & Sons, Inc. Page 7 of 37 Developmental Neurobiology
1 2 3 mesenchymal stromal cells (hMSC) were cultured according to instructions 4 5 6 provided by the supplier (Cambrex Corp). Vials containing cryogenically banked 7 8 hUTC or hMSC were thawed and plated at 5,000 cells/cm 2 in a T225 flask in 9 10 11 hUTC growth medium [low glucose DMEM (Gibco) containing 1% Penstrep 12 13 (Gibco) and 15% FBS (Hyclone) or hMSC (MSCGM™ (Lonza)] medium. After 2 14 15 days, hUTC or hMSC were rinsed in PBS, trypsinized and plated in transwell 16 17 5 18 permeable supportFor inserts (Costar)Peer at a concentratiReviewon of 10 cells/insert in 1.5ml 19 20 hUTC or hMSC medium. The next day, the cells were rinsed in warm PBS and 21 22 changed to serum free medium for 24 hours. For co culture, the inserts 23 24 25 containing the hUTC and hMSC were transferred into the wells containing the 26 27 hippocampal neurons on either 7 div or 16 div (see below). hUTC or hMSC were 28 29 tested for viability in the serum free medium using trypan blue exclusion and 30 31 32 methyl green positive staining in a parallel set of cultures. 33 34 35 36 37 Co-Culture and Treatments 38 39 Embryonic hippocampal cells were grown either for 7 or 16 days on polylysine 40 41 coated dishes prior to co culture with hUTC or hMSC in permeable transwell 42 43 44 inserts. The hippocampal cells were then fixed at the end of the 3 day co culture 45 46 period. rhBDNF (Peprotech) was used as a positive control at a concentration of 47 48 50 ng/ml. Chicken polyclonal BDNF Ab (Promega G1641) was used at a 49 50 51 concentration of 10 g/ml. IgY (Promega G1161) was used as a negative control 52 53 at a concentration of 10 g/ml. There were six treatment groups: IgY, BDNF Ab, 54 55 BDNF + IgY, BDNF + BDNF Ab, hUTC + IgY, hUTC + BDNF Ab. Hippocampal 56 57 58 59 60 7 John Wiley & Sons, Inc. Developmental Neurobiology Page 8 of 37
1 2 3 cultures (16 div) and hUTC were pretreated with IgY or BDNF Ab for 3 hours 4 5 6 prior to the addition of rhBDNF or co culture hUTC with the hippocampal 7 8 neurons. 9 10 11 12 13 Immunocytochemistry 14 15 At the end of the 3 day co culture period, hippocampal cells were fixed in 4% 16 17 18 paraformaldehydeFor or 15 min Peer and blocked Review with 2% Normal Goat Serum (NGS), 19 20 0.1% Triton X 100 in PBS (450 mM NaCl and 20 mM phosphate buffer, pH 7.4) 21 22 followed by 3 washes in PBS. Fixed cells were incubated in mouse anti 23 24 25 Microtubule Association Protein 2 (MAP2, 1:1000) (clone HM 2, Sigma) overnight 26 27 at 4ºC. After 3 washes in PBS, cultures were incubated in goat anti mouse 28 29 secondary antibodies conjugated to AlexaFluor 594 (1:1000) (Invitrogen) at room 30 31 32 temperature (RT) for 1h. The base of the dish was cut out and the cells were 33 34 cover slipped with Fluormount antifade reagent (SouthernBiotech). The images 35 36 37 were captured on a Zeiss Apotome microscope at 40X and analyzed using 38 39 Axiovision software. 40 41 42 43 44 Dendritic Morphology Analysis 45 46 The number of primary dendrites, total length of the primary dendrites, average 47 48 length of primary dendrites as well as number of secondary dendrites, total 49 50 51 length of secondary dendrites and average length of secondary dendrites were 52 53 analyzed using Axiovision software (Zeiss) in isolated cells. A primary dendrite 54 55 was considered any direct emanation from the cell body until it either ended or 56 57 58 59 60 8 John Wiley & Sons, Inc. Page 9 of 37 Developmental Neurobiology
1 2 3 branched off into secondary dendrites. A secondary dendrite was considered 4 5 6 anything branching off from a primary dendrite until it either ended or branched 7 8 off again into tertiary dendrites. 9 12 neurons per treatment condition were 9 10 11 counted for dendritic morphology. Each experiment was repeated 3 times. 12 13 14 15 SDS PAGE and Western Blot Analysis 16 17 18 Three days after Forco culture, Peer hippocampal Review neurons were solubilized rapidly in lysis 19 20 buffer (20 mM Tris pH 8, 0.5% Triton X 100, 0.5% SDS, protease inhibitor tablet, 21 22 1 mM phenylmethanesulfonylfluoride, and 0.5 mM vanadate). Protein from 6 23 24 25 wells/condition was pooled. Protein was concentrated using Amicon centricon 26 27 with a 10kD cutoff to 250 l. Protein content was determined with by BCA protein 28 29 assay (Pierce Chemical Co). Equal amount (50 g) of protein was loaded onto 4 30 31 32 12% gradient Tris Glycine or 12% NuPAGE Bis/Tris gel (Invitrogen) and 33 34 transferred to polyvinylidene difluoride filter Immobilon P transfer membranes 35 36 37 (Millipore Corp). Subsequently, the membranes were blocked with 5% milk plus 38 39 5% normal donkey serum in Tris Base Saline Solution plus 0.1% Tween 20 40 41 (TBST) for 1 hour. The membranes were then subjected to incubation overnight 42 43 44 at 4°C with antibodies against synaptotagmin (Chemicon 1:800) or PSD95 45 46 (Affinity Bioreagents MA1 046 1:2000) overnight at 4 oC followed by 3 washes in 47 48 TBST and anti mouse HRP conjugated IgG (1:5000) for 1 hour at RT. Proteins 49 50 51 were visualized by chemiluminescence using the Enhanced Chemiluminescence 52 53 (ECL) detection kit (NEN). Levels of the immunopositive bands were quantified 54 55 densitometrically using Quantity One version 4.2.1 software (Bio Rad) (Thakker 56 57 58 59 60 9 John Wiley & Sons, Inc. Developmental Neurobiology Page 10 of 37
1 2 3 Varia et al., 2001). The levels of Glyceraldehyde 3 phosphate dehydrogenase 4 5 6 (GAPDH) protein are used for normalization using GAPDH antibody for 1 hour at 7 8 room temperature (Biodesign, 1:1000). Each experiment was repeated 3 times. 9 10 11 12 ELISA for BDNF levels 13 14 2 15 Two different lots of hUTC were seeded seperately at 5,000 cells/cm in T75 16 17 flasks each containing 15 milliliters of growth medium, and cultured for 24 hours. 18 For Peer Review 19 The medium was changed to a serum free medium (DMEM low glucose (Gibco), 20 21 22 0.1% (w/v) bovine serum albumin (Sigma), penicillin (50 Units/milliliter) and 23 24 streptomycin (50 micrograms/milliliter, Gibco) for 8 hours. Conditioned serum 25 26 free medium was collected at the end of incubation by centrifugation at 14,000×g 27 28 29 for 5 minutes and stored at −20° C. To estimate the number of cells in each flask, 30 31 cells were washed with PBS and trypsinized. Cells were centrifuged at 150×g for 32 33 34 5 minutes. The supernatant was removed, and cells were resuspended in 1 35 36 milliliter medium. Cell number was estimated with a hemocytometer. BDNF was 37 38 measured using SearchLight Proteome Arrays (Pierce Biotechnology Inc.). Data 39 40 41 are expressed as picograms per milliliter per million cells. 42 43 44 45 Statistical analyses 46 47 48 Statview software was used for analysis of all data. Data were analyzed using 49 50 two tailed Student t test or ANOVA followed by Fishers PLSD post hoc test for 51 52 multiple comparisons. p < 0.05 is considered significant. 53 54 55 56 57 58 59 60 10 John Wiley & Sons, Inc. Page 11 of 37 Developmental Neurobiology
1 2 3 RESULTS 4 5 6 7 8 Effect of hUTC or hMSC on dendritic outgrowth 9 10 11 12 13 To investigate the effect of hUTC or hMSC on hippocampal dendritic growth 14 15 during an early phase of in vitro maturation, embryonic hippocampal cells were 16 17 18 grown for 7 daysFor on polylysine Peer coated dishesReview prior to co culture with hUTC or 19 20 hMSC in permeable transwell inserts. The viability of the hUTC or hMSC in the 21 22 hippocampal culture medium was confirmed using trypan blue exclusion as well 23 24 25 as methyl green positive staining in a parallel set of cultures and was greater 26 27 than 80% based on qualitative observations. The hippocampal cells were then 28 29 fixed 3 days after co culture. BDNF treatment (50 ng/ml) was used as a positive 30 31 32 control since it is known to promote dendritic differentiation. Culture sets that did 33 34 not exhibit a response to BDNF were not included in the analysis. Dendrites of 35 36 37 pyramidal shaped neurons were identified using MAP2 immunocytochemistry 38 39 which allows the distinction of dendrites and axons (see Fig. 1). The average 40 41 number of primary dendrites was increased by BDNF but not by hUTC or hMSC 42 43 44 relative to control. BDNF increased the total length of primary dendrites but 45 46 hUTC and hMSC had no effect on this parameter. BDNF did not increase the 47 48 average length of primary dendrites and neither did hUTC or hMSC. Because 49 50 51 there was no positive effect of hUTC or hMSC on any parameters for primary 52 53 neurites during the 7 10 div treatment, secondary dendrites were not quantitated. 54 55 56 57 58 59 60 11 John Wiley & Sons, Inc. Developmental Neurobiology Page 12 of 37
1 2 3 To explore the effect of hUTC or hMSC on hippocampal cell maturation 4 5 6 during a later phase of development, hippocampal cells were grown for 16 days 7 8 prior to treatment with BDNF or co culture with hUTC or hMSC cells and then 9 10 11 fixed 3 days later. Primary neurite number, total length and average length as 12 13 well as secondary neurite number, total length and average length were 14 15 quantitated (see Fig. 2). BDNF treatment resulted in a statistically significant 16 17 18 increase in all parametersFor forPeer primary dendrites. Review Hippocampal cells cultured with 19 20 hUTC or hMSC both demonstrated a significant increase in primary total length 21 22 and average number of primaries (p < 0.05, t test, n = 3). The effect on 23 24 25 secondary dendrites was quantitated and whereas BDNF increased secondary 26 27 total length and average length, there was no significant effect of soluble factors 28 29 from hUTC or hMSC on any parameters for secondary dendrites. Thus the effect 30 31 32 of factors from hUTC or hMSC appear to be strongest on primary dendrites at 33 34 later stages of development. 35 36 37 38 39 Effect of hUTC or hMSC on synaptogenesis 40 41 42 43 44 To determine whether the alterations in dendritic morphology by hUTC or hMSC 45 46 are associated with increased synapse formation, expression of synaptic markers 47 48 was analyzed using Western blot technique. Hippocampal cells were grown for 49 50 51 16 days then treated with with BDNF as a positive control or co cultured with 52 53 hUTC or hMSC for 3 days. The levels of the postsynaptic marker postsynaptic 54 55 density protein 95kD (PSD95) and the presynaptic synaptic vesicle marker 56 57 58 59 60 12 John Wiley & Sons, Inc. Page 13 of 37 Developmental Neurobiology
1 2 3 synaptotagmin were quantitated (see Fig. 3). All data are normalized to the 4 5 6 levels of GAPDH for each sample and expressed as a fold change relative to the 7 8 control for that set. There was a significant increase by BDNF in PSD95 as well 9 10 11 as synaptotagmin expression (p < 0.05, t test, n=3). Hippocampal cells co 12 13 cultured with hUTC showed significant increases in PSD95 and synaptotagmin 14 15 expression as well. Although hMSC treated cultures did show a trend to 16 17 18 increasing expressionFor of bothPeer the pre andReview postsynaptic markers, the changes 19 20 were not statistically significant. Therefore for the remaining experiments, we 21 22 focused our studies on the effect of hUTC on hippocampal neurons. These 23 24 25 findings suggest that soluble factors from hUTC promote expression of proteins 26 27 involved in the formation of synapses and synaptic transmission. 28 29 30 31 32 BDNF release from hUTC 33 34 35 36 37 BDNF secretion was measured over eight hours following 24 hrs of culture in two 38 39 different lots of hUTC and was found to be 165.7 pg/ml/million cells and 388.6 40 41 pg/ml/million cells respectively (See Fig. 4). These findings suggest that BDNF is 42 43 44 released in detectable levels from hUTC. 45 46 47 48 Effect of BDNF blocking Ab on dendrite differentition by hUTC 49 50 51 52 53 Hippocampal cells were grown for 16 days prior to treatment with BDNF plus 54 55 BDNF Ab or IgY as a negative control and then fixed at the end of the 3 day co 56 57 58 59 60 13 John Wiley & Sons, Inc. Developmental Neurobiology Page 14 of 37
1 2 3 culture period. Dendrites were identified using MAP2 immunocytochemistry. 4 5 6 Primary neurite number, total length and average length as well as secondary 7 8 number, total length and average length were quantitated (see Fig. 5). When the 9 10 11 data are normalized to the IgY control for each set, there is a statistically 12 13 significant increase by BDNF + IgY of dendritic number, total length for both 14 15 primary and secondary dendrites and average length of primaries (p < 0.05, 16 17 18 ANOVA, Fisher’sFor post hoc Peertest, n=3). ThereReview was no significant effect of BDNF 19 20 Ab by itself on any of the parameters measured relative to IgY indicating that the 21 22 BDNF Ab did not have deleterious effects on the hippocampal cells. The BDNF + 23 24 25 Ab set was significantly reduced relative to the BDNF + IgY groups with respect 26 27 to number of dendrites for primaries and secondaries, total dendritic length for 28 29 primaries and secondaries, and average dendritic length for primaries. The only 30 31 32 case in which the BDNF + Ab did not reduce the effect of BDNF was average 33 34 length of secondary dendrites which also had no significant effect of BDNF + IgY 35 36 37 relative to IgY. Therefore the BDNF Ab blocked the BDNF effect on all of the 38 39 parameters that were enhanced by exogenous BDNF. 40 41 42 43 44 The effect of the BDNF blocking Ab on the effect of hUTC was then 45 46 examined by co culturing hippocampal cells from 16 19 div in the presence of 47 48 hUTC plus BDNF Ab or IgY as a negative control. hUTC + IgY had a significant 49 50 51 effect relative to IgY on enhancing total length for both primary and secondary 52 53 dendrites, average length for both primary and secondary dendrites, but not for 54 55 dendritic number of either primary or secondary dendrites. hUTC + BDNF Ab 56 57 58 59 60 14 John Wiley & Sons, Inc. Page 15 of 37 Developmental Neurobiology
1 2 3 were significantly different from hUTC + IgY for total length of both primary and 4 5 6 secondary dendrites. There was a trend toward shorter average length on 7 8 primary and secondary dendrites in the hUTC + Ab relative to hUTC + IgY 9 10 11 groups but the effect was not significant (Figure 3). Finally, there was a 12 13 significant reduction of the number of primary and secondary dendrites in the 14 15 hUTC + Ab group relative to hUTC + IgY (Figure 4). Therefore, the positive 16 17 18 effects of hUTC onFor hippocampal Peer neurite differentiatReviewion can be blocked, in part, by 19 20 BDNF neutralizing antibody. 21 22 23 24 25 DISCUSSION 26 27 28 29 Dendritic morphology is known to be plastic and responsive to both soluble and 30 31 32 membrane bound factors in the environment (Horch, 2004). In our study, 33 34 hippocampal neurons at 16 19 div exhibited an increase in the number of primary 35 36 37 dendrites as well as total dendritic length with no change in average dendritic 38 39 length relative to control when co cultured with hUTC or hMSC for 3 days. 40 41 Therefore soluble factors from hUTC or hMSC appeared to be promoting primary 42 43 44 dendrite initiation but not dendritic extension. Although factors released by 45 46 hUTC or hMSC had an effect of hippocampal neurons at 16 19 div, this same 47 48 effect was not observed on hippocampal neurons at 7 10 div. One possible 49 50 51 explanation could be that younger hippocampal cultures require higher levels of 52 53 secreted factors than the older cultures to affect morphology so that only the 16 54 55 19 div hippocampal cells were able to respond to the level of factors secreted by 56 57 58 59 60 15 John Wiley & Sons, Inc. Developmental Neurobiology Page 16 of 37
1 2 3 hUTC or hMSC. Alternatively, rodent hippocampal cells may need to express 4 5 6 certain receptors or molecules at a sufficient level to be able to respond to the 7 8 factors released by the hUTC or hMSC. Those receptors may not yet be present 9 10 11 at sufficient levels in the younger cultures. By 16 19 div, however, the 12 13 hippocampal neurons may express those receptors at sufficient levels to respond 14 15 to the factors secreted by hUTC or hMSC. TrkB is expressed as early as 2 days 16 17 18 in vitro (Marsh etFor al., 1993) Peer although it isReview not known at what point maximal TrkB 19 20 expression and activation of signaling cascades is reached. Future studies 21 22 examining a developmental time course of TrkB expression levels and second 23 24 25 messenger activation will yield insight whether receptor expression is a 26 27 contributing factor to the temporal response to BDNF in vitro. The fact that 28 29 positive control consisting of exogenous rhBDNF had an effect on the rodent 30 31 32 hippocampal neurons at the earlier stage of development (7 10 div) and had 33 34 more robust effects at later stages than the hUTC or hMSC may be due to the 35 36 37 fact that exogenous BDNF was added to the cultures at higher concentration 38 39 than the BDNF secreted from hUTC based on results from the ELISA assay. 40 41 Overall, our data suggest that hUTC as well as hMSC promoted dendritic 42 43 44 differentiation of rodent hippocampal cells in co culture conditions. 45 46 47 48 To measure synaptogenesis more directly we examined the expression of 49 50 51 synaptic markers. We chose PSD95 as a postsynaptic marker because it is 52 53 involved in anchoring synaptic proteins in the postsynaptic density. The effect of 54 55 beta Estradiol on PSD95 expression and synaptogenesis in the hippocampus 56 57 58 59 60 16 John Wiley & Sons, Inc. Page 17 of 37 Developmental Neurobiology
1 2 3 has also been linked to BDNF (Sato et al., 2007). Synaptotagmin, a membrane 4 5 6 trafficking protein in synaptic vesicles, was selected as a presynaptic marker in 7 8 this study because it is thought to modulate synaptic function in part by regulating 9 10 11 BDNF release (Dean et al., 2009) and BDNF mutant mice have impairments in 12 13 synaptic vesicle docking and protein distribution (Pozzo Miller et al., 1999). 14 15 When co cultured with hUTC, an increase in expression of both pre and 16 17 18 postsynaptic markersFor over Peercontrol levels Reviewwere observed, similar to the effects of 19 20 rhBDNF. Co culture with hMSC also resulted in a trend towards enhanced 21 22 synaptic marker expression as compared to controls. However, the change in 23 24 25 PSD95 expression observed in the hMSC co culture vs. controls was not 26 27 significant. This finding may mean that hMSC had an effect on the morphological 28 29 aspects of neuronal maturation of rodent hippocampal cells but not on the 30 31 32 molecular changes underlying synaptogenesis that require protein synthesis. 33 34 The observed change in both the dendritic arborization as well as an increase in 35 36 37 the pre and postsynaptic markers of the hippocampal neurons by hUTC suggests 38 39 that hUTC has both morphological as well as molecular effects on the 40 41 hippocampal cells. We chose to focus our studies on hUTC because of its more 42 43 44 reliable effects on synaptic marker expression. Our findings therefore suggest 45 46 that there may be more synapses being formed in the hippocampal neurons co 47 48 cultured with hUTC versus hMSC implying that the connection between the 49 50 51 neurons is strengthened by a factor(s) released by hUTC. 52 53 54 55 56 57 58 59 60 17 John Wiley & Sons, Inc. Developmental Neurobiology Page 18 of 37
1 2 3 The detection of BDNF in the conditioned media from hUTC indicates that 4 5 6 the hUTC secrete BDNF. Futhermore, a BDNF functional blocking antibody 7 8 significantly inhibited the effects of hUTC enhancement on dendritic morphology, 9 10 11 suggesting that BDNF contributed to the effects of hUTC on dendritic 12 13 development of rodent hippocampal cells. BDNF Ab has been used to 14 15 demonstrate that endogenous BDNF is responsible for a number of changes in 16 17 18 neuronal functionFor (Koyama Peeret al., 2004; AlonsoReview et al., 2005; Massey et al., 2006; 19 20 Pugh et al., 2006; Sanchez et al., 2006). To study the effects of BDNF on 21 22 hippocampal neurons, we typically treat the neurons with 50ng/ml recombinant 23 24 25 human BDNF (Thakker Varia et al., 2001). Human umbilical tissue derived cells 26 27 when cultured alone secrete low levels of BDNF. The finding that the actions of 28 29 hUTC on dendritic morphology can be partially blocked with a neutralizing 30 31 32 antibody to BDNF demonstrates that hUTC derived BDNF is secreted at 33 34 sufficient levels to play a role in dendritic differentiation in this in vitro model. 35 36 37 Although BDNF antibody inhibited the number of and total dendritic length 38 39 induced by hUTC co culture, the average dendritic length is not significantly 40 41 inhibited. Thus, our data do not rule out the possibility that there may be other 42 43 44 secreted factors that also contribute to the response of the hippocampal cells to 45 46 the hUTC which could be tested in future experiments. 47 48 49 50 51 BDNF has been shown to act as both an autocrine and paracrine 52 53 molecule by affecting dendritic branching and growth in pyramidal cells secreting 54 55 BDNF as well as neighboring neurons (Horch and Katz, 2002; Horch, 2004) thus 56 57 58 59 60 18 John Wiley & Sons, Inc. Page 19 of 37 Developmental Neurobiology
1 2 3 indicating it can work at both proximal and longer distances when secreted. The 4 5 6 effect of distance of the hUTC or hMSC from the hippocampal neurons could be 7 8 examined in the future. Furthermore, while this study focused on the effects of 9 10 11 cells on excitatory pyramidal shaped neurons, BDNF also affects dendritic 12 13 branching of inhibitory interneurons (Vicario Abejon et al., 1998; Jin et al., 2003; 14 15 Wirth et al., 2003; Woo et al., 2005) and dentate granule cells (Patel and 16 17 18 McNamara, 1995;For Danzer Peer et al., 2002), Review therefore, future studies could explore 19 20 the effects of hUTC or hMSC on other hippocampal neuronal subtypes. 21 22 23 24 25 Previous studies by Koh have shown that intracerebral implantation of 26 27 stem cells two weeks after induction of focal ischemia reduced damage and 28 29 improved neurological function in a rodent model (Koh et al., 2008). 30 31 32 Furthermore, intravenous administration of hUTC following induction of stroke 33 34 improves sensori motor function without altering the lesion volume with evidence 35 36 37 of neurogenesis, and increased synaptic and vascular densities. In that study, 38 39 treatment with hUTC increased synaptophysin immunoreactivity at the ischemic 40 41 boundary area (Zhang et al., 2011) consistent with our in vitro data indicating 42 43 44 increased synaptotagmin expression by hippocampal neurons. The effect of cell 45 46 therapy in stroke models has been attributed to neurotrophins (Li et al., 2002; 47 48 Yasuhara et al., 2006). Specifically, BDNF has been shown to induce 49 50 51 neurogenesis, synaptogenesis and angiogenesis after stroke (Schabitz et al., 52 53 2004) and to activate anti apoptotic pathways (Schabitz et al., 2000). In 54 55 conclusion, the positive effect of hUTC on rodent hippocampal neurite 56 57 58 59 60 19 John Wiley & Sons, Inc. Developmental Neurobiology Page 20 of 37
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1 2 3 Zhang L, Li Y, Zhang C, Chopp M, Gosiewska A, Hong K. 2011. Delayed 4 5 6 administration of human umbilical tissue derived cells improved 7 8 neurological functional recovery in a rodent model of focal ischemia. 9 10 11 Stroke; a journal of cerebral circulation 42:1437 1444. 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 29 John Wiley & Sons, Inc. Developmental Neurobiology Page 30 of 37
1 2 3 FIGURE LEGENDS 4 5 6 7 8 Figure 1 . hUTC or hMSC do not affect dendrite growth in hippocampal cultures 9 10 11 7 – 10 div. A: Dendrites were identified using MAP2 immunocytochemistry 12 13 followed by quantitation with Axiovision software. The average number of 14 15 primary dendrites was increased by BDNF but not by hUTC or hMSC relative to 16 17 18 control (vehicle).For B: BDNF Peer increased theReview total length of primary dendrites but 19 20 hUTC or hMSC had no effect on this parameter. C: BDNF did not increase the 21 22 average length of primary dendrites relative to control and neither did hUTC or 23 24 25 hMSC. Because there was no enhancement of any primary neurite parameters 26 27 measured for hUTC or hMSC, secondary neurites were not quantitated. n = 9 28 29 12 cells/group and the experiment was repeated 3 times. Error bars represent 30 31 32 SEM. * indicates p < 0.05, unpaired two tailed Students’s t test relative to 33 34 control. 35 36 37 38 39 Figure 2 . hUTC or hMSC promote dendrite maturation in hippocampal cultures 40 41 16 – 19 div. A – D: Representative images of MAP2 positive cells in hippocampal 42 43 44 cultures treated with A: Control (vehicle), B: BDNF, C: hUTC, D: hMSC. Images 45 46 of isolated cells were acquired on Zeiss Apotome microscope at 40X. Scale bar = 47 48 75 m. E G: Quantitation of dendritic parameters using Axiovision software 49 50 51 expressed as a fold change relative to control (vehicle). E: The average number 52 53 of primaries was increased by BDNF, hUTC or hMSC but the average number of 54 55 secondaries was only increased by BDNF. F: The total length of primaries was 56 57 58 59 60 30 John Wiley & Sons, Inc. Page 31 of 37 Developmental Neurobiology
1 2 3 increased by BDNF, hUTC or hMSC but total length of secondaries was not 4 5 6 significantly affected by any of the conditions. G: BDNF enhanced the average 7 8 length of primaries but hUTC or hMSC had no significant effect and there was no 9 10 11 effect of any of the conditions on secondary average dendritic length. n = 9 12 12 13 cells/group and the experiment was repeated 3 times. Error bars represent SEM. 14 15 * indicates p > 0.05, unpaired two tailed Students’s t test relative to control. 16 17 18 For Peer Review 19 20 Figure 3 . hUTC promote synaptogenesis in hippocampal cultures 16 – 19 div. A: 21 22 Representative Western blot of PSD95 and synaptotagmin protein expression in 23 24 25 hippocampal neurons co cultured with control (vehicle), BDNF, hUTC or hMSC. 26 27 Each lane represents protein harvested from 8 culture dishes. B: Quantitation of 28 29 protein levels indicates that BDNF or hUTC significantly upregulate PSD95 and 30 31 32 synaptotagmin expression. hMSCs show a trend to increased synapse marker 33 34 expression. The bars represent average protein levels relative to control (vehicle) 35 36 37 ± SEM (n = 3). All data are first normalized to GAPDH expression which controls 38 39 for protein loading. * indicates p > 0.05, unpaired two tailed Students’s t test 40 41 relative to control. 42 43 44 45 46 Figure 4. hUTC secrete BDNF. ELISA was used to measure BDNF levels 47 48 secreted from hUTC over 8 hours in serum free medium (SFM). Two different 49 50 51 lots of hUTC were tested. Bars represent the pg BDNF detected per ml per 1 52 53 million hUTC. No BDNF was present in the negative control SFM (ND = not 54 55 detected). 56 57 58 59 60 31 John Wiley & Sons, Inc. Developmental Neurobiology Page 32 of 37
1 2 3 4 5 6 Figure 5. The effect of hUTC on dendrite maturation requires BDNF. A – F: 7 8 Representative images of MAP2 positive cells in hippocampal cultures treated 9 10 11 from 16 – 19 div with A: IgY, B: BDNF Ab, C: BDNF+IgY, D: BDNF+BDNF Ab, E: 12 13 hUTC+IgY and F: hUTC+BDNF Ab. Images of isolated cells were acquired on 14 15 Zeiss Apotome microscope at 40X. Scale bar = 75 m. G I: Quantitation of 16 17 18 dendritic parametersFor using Peer Axiovision software Review which is then normalized to the 19 20 change by IgY treatment. G: The BDNF blocking Ab significantly reduced the 21 22 number of primary and secondary dendrites when hippocampal cultures were 23 24 25 pre treated prior to the addition of BDNF or hUTC. H: BDNF Ab blocked the 26 27 effect of BDNF and hUTC on total dendritic length for both primary and 28 29 secondary dendrites. I: BDNF Ab blocked the effect of BDNF on average 30 31 32 dendritic length and there was a trend towards reduced dendritic length by hUTC 33 34 in the presence of the BDNF Ab. Bars represent average of 3 experiments, 9 12 35 36 37 cells quantitated per experiment. Error bars are SEM. Statistical analysis 38 39 consists of ANOVA followed by Fisher's post hoc testing with p < 0.05 considered 40 41 significant. * indicates BDNF + IgY or hUTC + IgY is significantly different from 42 43 44 IgY. + indicates that BDNF + antibody (Ab) is significantly different from BDNF + 45 46 IgY or that hUTC + Ab is significantly different from Q+ IgY. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 32 John Wiley & Sons, Inc. Page 33 of 37 Developmental Neurobiology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 Figure 1. hUTC or hMSC do not affect dendrite growth in hippocampal cultures 7 – 10 div. A: Dendrites 25 were identified using MAP2 immunocytochemistry followed by quantitation with Axiovision software. The 26 average number of primary dendrites was increased by BDNF but not by hUTC or hMSC relative to control 27 (vehicle). B: BDNF increased the total length of primary dendrites but hUTC or hMSC had no effect on this 28 parameter. C: BDNF did not increase the average length of primary dendrites relative to control and neither 29 did hUTC or hMSC. Because there was no enhancement of any primary neurite parameters measured for hUTC or hMSC, secondary neurites were not quantitated. n = 9 - 12 cells/group and the experiment was 30 repeated 3 times. Error bars represent SEM. * indicates p < 0.05, unpaired two tailed Students’s t-test 31 relative to control. 32 1625x812mm (72 x 72 DPI) 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Developmental Neurobiology Page 34 of 37
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 Figure 2. hUTC or hMSC promote dendrite maturation in hippocampal cultures 16 – 19 div. A – D: 28 Representative images of MAP2 positive cells in hippocampal cultures treated with A: Control (vehicle), B: BDNF, C: hUTC, D: hMSC. Images of isolated cells were acquired on Zeiss Apotome microscope at 40X. 29 Scale bar = 75 m. E G: Quantitation of dendritic parameters using Axiovision software expressed as a fold 30 change relative to control (vehicle). E: The average number of primaries was increased by BDNF, hUTC or 31 hMSC but the average number of secondaries was only increased by BDNF. F: The total length of primaries 32 was increased by BDNF, hUTC or hMSC but total length of secondaries was not significantly affected by any 33 of the conditions. G: BDNF enhanced the average length of primaries but hUTC or hMSC had no significant 34 effect and there was no effect of any of the conditions on secondary average dendritic length. n = 9 12 35 cells/group and the experiment was repeated 3 times. Error bars represent SEM. * indicates p > 0.05, 36 unpaired two tailed Students’s t test relative to control. 155x90mm (300 x 300 DPI) 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 35 of 37 Developmental Neurobiology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 3. hUTC promote synaptogenesis in hippocampal cultures 16 – 19 div. A: Representative Western blot of PSD95 and synaptotagmin protein expression in hippocampal neurons co-cultured with control 48 (vehicle), BDNF, hUTC or hMSC. Each lane represents protein harvested from 8 culture dishes. B: 49 Quantitation of protein levels indicates that BDNF or hUTC significantly upregulate PSD95 and 50 synaptotagmin expression. hMSCs show a trend to increased synapse marker expression. The bars 51 represent average protein levels relative to control (vehicle) ± SEM (n = 3). All data are first normalized to 52 GAPDH expression which controls for protein loading. * indicates p > 0.05, unpaired two tailed Students’s t- 53 test relative to control. 54 133x220mm (300 x 300 DPI) 55 56 57 58 59 60 John Wiley & Sons, Inc. Developmental Neurobiology Page 36 of 37
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Figure 4. hUTC secrete BDNF. ELISA was used to measure BDNF levels secreted from hUTC over 8 hours in 38 serum free medium (SFM). Two different lots of hUTC were tested. Bars represent the pg BDNF detected 39 per ml per 1 million hUTC. No BDNF was present in the negative control SFM (ND = not detected). 89x80mm (300 x 300 DPI) 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 37 of 37 Developmental Neurobiology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Figure 5. The effect of hUTC on dendrite maturation requires BDNF. A – F: Representative images of MAP2 positive cells in hippocampal cultures treated from 16 – 19 div with A: IgY, B: BDNF Ab, C: BDNF+IgY, D: 47 BDNF+BDNF Ab, E: hUTC+IgY and F: hUTC+BDNF Ab. Images of isolated cells were acquired on Zeiss 48 Apotome microscope at 40X. Scale bar = 75 m. G I: Quantitation of dendritic parameters using 49 Axiovision software which is then normalized to the change by IgY treatment. G: The BDNF blocking Ab 50 significantly reduced the number of primary and secondary dendrites when hippocampal cultures were pre 51 treated prior to the addition of BDNF or hUTC. H: BDNF Ab blocked the effect of BDNF and hUTC on total 52 dendritic length for both primary and secondary dendrites. I: BDNF Ab blocked the effect of BDNF on 53 average dendritic length and there was a trend towards reduced dendritic length by hUTC in the presence of 54 the BDNF Ab. Bars represent average of 3 experiments, 9 12 cells quantitated per experiment. Error bars are SEM. Statistical analysis consists of ANOVA followed by Fisher's post hoc testing with p < 0.05 55 considered significant. * indicates BDNF + IgY or hUTC + IgY is significantly different from IgY. + indicates 56 that BDNF + antibody (Ab) is significantly different from BDNF + IgY or that hUTC + Ab is significantly 57 different from Q+ IgY. 58 59 60 John Wiley & Sons, Inc. Developmental Neurobiology Page 38 of 37
1 2 3 263x308mm (300 x 300 DPI) 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc.