Neurochem Res DOI 10.1007/s11064-017-2401-y

ORIGINAL PAPER

Human Umbilical Cord Mesenchymal Stem Cells Overexpressing Nerve Growth Factor Ameliorate Diabetic Cystopathy in Rats Wu WenBo1 · Zhang Fei2 · Du YiHeng1 · Wang Wei1 · Yan TingMang1 · Zhou WenHao1 · Liu QianRu3 · Liu HaiTao1 

Received: 7 February 2017 / Revised: 10 August 2017 / Accepted: 11 September 2017 © Springer Science+Business Media, LLC 2017

Abstract  Diabetic cystopathy is a common complication of voiding disorders in diabetes mellitus. Neuropathy and bladder remodeling underlie the lack of efficacy of pharmacological and surgical treatments. Previous studies have shown that decreased levels of nerve growth factor (NGF) are closely associated with disease progression. Besides, application of human umbilical cord mesenchymal stem cells (hUC-MSCs) is also considered a promising therapeutic strategy for treatment of diabetic neuropathy. In our study, we determine the therapeutic efficacy and mechanisms of hUC-MSCs which transfected with NGF geen in ameliorating diabetic cystopathy for the first time. We transducted hUC-MSCs with NGF-expressing lentivirus so that the hUC-MSCs can express NGF efficiently, then the NGFexpressing hUC-MSCs were intrathecally administrated in L6–S1 spinal cord of diabetic rats 3 days after induced by streptozotocin. Nine weeks later, the level of neurotrophins and voiding function of bladder were detected. Results show that improvements in voiding function were related to the Wu WenBo and Zhang Fei have contributed equally to this work and Zhang Fei should be considered as co-first author. Electronic supplementary material  The online version of this article (doi:10.1007/s11064-017-2401-y) contains supplementary material, which is available to authorized users. * Liu HaiTao [email protected] 1



Department of Urology, Shanghai General Hospital (Shanghai Peoples Hosp 1), Shanghai JiaoTong University School of Medicine, 100 Haining Rd, Shanghai 200080, China

2



Department of Urology, The Affiliated Hospital of School of Medicine of NingBo University, Ningbo, China

3

QUFU Normal University, Jining, Shandong, China



neurotrophins and cytokines released by the intrathecally transplanted hUC-MSCs. In addition, the hUC-MSCs also differentiated into neurons and astrocytes within the spinal cord in rats. These two mechanisms play a combined role in neural regeneration and the amelioration of the symptoms of diabetic cystopathy. Keywords  Human umbilical cord mesenchymal stem cells (hUC-MSCs) · Diabetic cystopathy · Nerve growth factor (NGF) · Intrathecal administration · Functional study

Introduction The number of individuals with diabetes mellitus (DM) is increasing rapidly; up to 366 million cases are expected in the next 15 years [1]. Diabetic cystopathy is a common complication of DM and is characterized by decreased bladder contractility and sensation and increased bladder capacity, which leads to an increase in residual urine, urinary retention, and overflow incontinence [2, 3]. Diabetic cystopathy remains an intractable problem for which pharmacological and surgical interventions are largely ineffective [4, 5]. Extensive evidence indicates that the stem cell transplantation is a therapeutic strategy with potential to treat neurological disorders such as Alzheimer’s disease [6] and other neurodegenerative diseases [24] as well as epilepsy [7], spinal cord injury (SCI) [22], and multiple sclerosis [23]. Mesenchymal stem cells (MSCs) are multipotent stem cells that are present in several tissues and can be expanded in vitro, and they have a high capacity for self-renewal and multi-differentiation potential [8]. MSCs are also nonimmunogenic, and therefore do not elicit a proliferative response from endogenous lymphocytes [9]. Additionally, under defined culture conditions, MSCs can be induced to

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differentiate into neuronal, muscle, and fat cells, osteoblasts, and chondrocytes. Human umbilical cord mesenchymal stem cells (hUCMSCs) derived from Wharton’s jelly are morphologically and immunophenotypically similar to MSCs isolated from bone marrow [27] and have strong proliferative and differentiation potential [10]. These cells are obtained by noninvasive procedures, thus avoiding ethical restrictions, and are therefore considered an ideal source of human-derived MSCs for use in clinical studies and experimental research [11]. hUC-MSC transplantation has been reported to be efficacious against neurological diseases in animal models such as ischemic stroke [18, 19], Parkinson’s disease [20], and SCI [21]. Previous studies have shown a link between deficiency in retrograde axonal transport of nerve growth factor (NGF) released by target organs to sensory pathways and diabetic neuropathy [13, 14]. NGF levels were found to decrease over time in the L6–S1 spinal nerve center of bladder, leading to bladder voiding dysfunction [12]. HSV vector-mediated NGF treatment in animal models of diabetes has shown that gene therapy can increase NGF expression in the bladder and its afferent pathways and improve bladder function [15, 16]. These studies demonstrate that transgenic and cell engineering technologies can be applied to the treatment of urinary system dysfunction. To develop a novel, effective therapy for diabetic cystopathy based on hUC-MSCs, NGF-expressing hUC-MSCs were established in the current study, and their therapeutic efficacy and mechanisms were investigated in streptozotocininduced diabetic rats in vivo.

Materials and Methods hUC‑MSC Isolation and Identification The study protocol was approved by Shanghai General Hospital Ethics Committee. Three human UC samples were obtained from three healthy mothers at Shanghai General Hospital of Shanghai Jiaotong University. Cord vessels were removed to prevent endothelial cell contamination and cords were rinsed several times in phosphatebuffered saline (PBS), then cut into small pieces (approximately 1–2 mm3) that were immediately placed in 6-well plates in 2  ml of Dulbecco’s modified Eagle medium (DMEM) containing 15% fetal bovine serum (FBS; Millipore, Darmstadt, Germany) and 100 U/ml penicillin/streptomycin for culture expansion. After 3 days, non-adherent cells were discarded by washing with PBS; adherent cells were defined as passage (P0). hUC-MSCs were subcultured to P3 before use in experiments. Third-generation

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hUC-MSCs were harvested to evaluate osteogenic and adipogenic differentiation by Alizarin Red and Oil Red O staining, respectively. Flow Cytometry Expression of MSC markers was detected by flow cytometry on a FACSAria instrument (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, 1 × 105 P3 cells were characterized by labeling with antibodies against cluster of differentiation (CD) CD31, CD34, and CD45, and the human leukocyte antigen (HLA)-DR markers CD44, CD29, CD90, and CD105 (BD Biosciences). Transduction of hU‑MSCs with Lentivirus and Enzyme‑Linked Immunosorbent Assay The lentivirus purchased from Genechem Co. (Shanghai, China) contained NGF and green fluorescent protein (GFP) genes. hUC-MSCs were trypsinized and seeded at a density of 5 × 104 cells in 0.5 ml of DMEM supplemented with 10% FBS in 12-well plates. The lentivirus was immediately added at a multiplicity of infection of 50 in the presence of 8 μg/ ml polybrene (Sigma-Aldrich, St. Louis, MO, USA). After 12 h, the medium was replaced with 1 ml of DMEM-F12 supplemented with 10% FBS. To quantify the percentage of successfully transduced hUC-MSCs, we randomly selected ten high-power fields and count the infection rate. The NGF protein concentration in the culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA) using a kit (Bluegen Systems, Beijing, China) according to the manufacturer’s instructions after washing of the cells with PBS and addition of fresh DMEM-F12 supplemented with 0.1% FBS. Rat Model of Diabetes The experiment was approved by Ethical Committee on Animal Experiment Committee of Shanghai Jiao Tong University School of Medicine. Sprague–Dawley rats (n = 90; 200–210 g) were purchased from Shanghai Silaike Experimental Animals Co. (QZ:SCXK; Shanghai, China). The 80 rats received intraperitoneal injections of 60 mg/kg streptozotocin (STZ), whereas the latter received the same volume of citric acid buffer. Fasted blood glucose was analyzed 72 h after STZ injection; a level > 16.7 mmol/l was considered to indicate successful induction of diabetes. The number of diabetic rats was 67, the ratio is 83.7%. And then we chose 60 diabetic rats as diabetic group (n = 60) and ten untreated rats as control groups (n = 10).

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hUC‑MSC Transplantation Rats were grouped in two cohorts: normal control(N, n = 10) and diabetic (DM, n = 60). The diabetic group was divided into PBS control (con), hUC-MSC treatment (hUC-MSCs), NGF-hUC-MSC treatment (NGF-hUC-MSCs), and NGFlentivirus-only treatment (NGF-Lv) groups (each group n = 15). The groups received intrathecal injection of PBS, hUC-MSCs, NGF-hUC-MSCs and NGF-lentivirus, respectively, as previously described [25]. Animals were then anesthetized with isoflurane and a small (1 cm) longitudinal incision was made over the L6 to S1 spinous processes. A 25-gauge lumbar puncture needle (BD Biosciences) was inserted into the spinal canal at the L6 to S1 level. Correct placement of the needle in the lumbar subdural space was confirmed based on loss of resistance from the moment of entry, tail flicking, and the presence of cerebrospinal fluid in the needle hub. Then, hUC-MSCs or NGF-hUC-MSCs (1 × 106) and 5 × 107 NGF-lentivirus (moi = 50) diluted in 50 μl PBS were injected into spinal canal over 30 s. The entire procedure lasted 10 min. Cystometry Functional studies were conducted in all groups 9 weeks after cell transplantation. Cystometry was performed as previously described [30]. General anesthesia was induced with isoflurane, a polyethylene-90 catheter implanted into the bladder 24 h before the procedure. The bladder catheter was connected with a pressure transducer and infusion pump. Filling the bladder via the catheter with room-temperature PBS at 0.1 ml/min while simultaneously recording bladder pressure by Medlab-U/4C501 software. The micturition interval (s), urine volume per void (ml), maximum voiding pressure (cm ­H2O), and residual volume (ml) were recorded after 30 min. Immunohistochemistry and Hematoxylin–Eosin Staining After the functional study, at 9 weeks after transplantation, five rats in each group were sacrificed after cystometry for histological study. Tissue samples of the bladder were dehydrated by ethanol and embedded in paraffin wax, followed by histologic sectioning (10 μm thick) and hematoxylin–eosin staining. In addtion, tissue sections (10 μm thick) of the L6–S1 spinal cord were incubated overnight (12 h) at 4 °C with primary antibodies against the following proteins: NGF (1:100; Proteintech, Rosemont, IL, USA), brain-derived neurotrophic factor (BDNF, 1:100), and neurotrophin (NT-3, 1:100)(all from Proteintech, Rosemont, IL, USA). Sections were washed three times with PBS for 5 min and then incubated for 24 h with the appropriate secondary

antibody. After three 5-min washes with PBS, horseradish peroxidase (HRP)-avidin–biotin complex was applied for 15 min at room temperature and sections were then stained with diaminobenzidine, washed, mounted, and visualized by light microscopy (CX41; Olympus, Tokyo, Japan). The mean integral optical density of nine randomly selected sections was determined with Image Pro Plus software (Media Cybernetics, Rockville, MD, USA). Immunofluorescence NGF-hUC-MSCs expressed GFP protein; therefore, we were able to track transplanted hUC-MSCs by their green fluorescence. We were also able to demonstrate the differentiation ability as follows. Six weeks after transplantation, five rats in the NGF-hUC-MSC group and each other groups were sacrificed and spinal tissue in L6–S1 was made into cryostat section and detect the survival of transplanted hUC-MSCs. And in positive-NGF-hUC-MSC group we also detect the differentiation of transplanted hUC-MSCs. Sections (10 μm) of the L6–S1 spinal were incubated with rabbit anti-glial fibrillary acidic protein (GFAP) antibody (Proteintech) and rabbit anti-NeuN antibody (Proteintech) overnight (12 h) at 4 °C. Sections were washed three times for 5 min and then incubated for 24 h with rhodamine-labeled mouse anti-rabbit IgG, and visualized by epifluorescence microscopy (Leica, Wetzlar, Germany). Western Blot Analysis Five rats in each group were sacrificed after cystometry test. The spinal cord in L6–S1 was dissected from each rat on an ice-chilled plate. The tissue was homogenized with a 1.0-ml Dounce grinder (Wheaton, Millville, NJ, USA) and type B pestle in a 10:1 w/v buffer containing 10 mM Tris (pH 7.4), 10 mM EGTA, 250 mM sucrose, 2 μg/ml aprotinin, 5 μg/ml leupeptin, 2 μg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. Protein concentrations of tissue lysates were determined with a Bio-Rad protein assay kit. Equal amounts (20 μg) of protein were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Little Chalfont, UK), which were blocked with 5% skim milk and incubated with antibodies against NGF, BDNF and NT-3 (1:1000) (all from Proteintech, Rosemont, IL, USA). Immunoreactivity was detected using an HRP-conjugated anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA, USA). β-Actin was used as a loading control. Statistical Analysis Data are expressed as the mean ± standard deviation (SD) of experiments performed in triplicate and were

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analyzed with SPSS v.18.0 software (SPSS Inc., Chicago, IL, USA). Differences between groups were evaluated by one-way analysis of variance, followed by the least-squares difference test for homogeneity of variance or Dunnett’s T3 test for heterogeneity of variance as posthoc multiple comparisons. P < 0.05 was considered statistically significant.

Results

Fig.  1  a Surface marker expression in hUC-MSCs at P3. hUC-MSCs were negative for CD31, CD34, CD45, and HLA-DR and positive for CD29, CD44, CD90, and CD105, confirming that the cells were MSCs and not hematopoietic or endothelial cells. b Alizarin Red S staining of hUC-MSCs shows calcium deposits stained bright orange-

red in osteocytes on day 21. c Oil Red O staining of hUC-MSCs shows intracellular lipids in adipocytes stained bright red on day 21 (Both MSC cultures were stained after culturing in specific osteogenic or adipogenic media)

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Characteristics of hUC‑MSCs All experiments were performed on the monolayer cultures of hUC-MSC in  vitro, hUC-MSCs had a fibroblast-like appearance, and flow cytometry analysis revealed CD31-CD34-CD45-HLA-DR- and CD29+CD90+CD44+CD105+phenotypes (Fig. 1a). The proportion of cells expressing CD29, CD90, CD44, and

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CD105 was 100.0, 100.0, 99.9, and 98.8%, respectively. CD31, CD34, CD45, and HLA-DR were expressed in few cells (0.032, 0.00, 0.00, and 0.00%, respectively). hUC-MSCs were stained with Alizarin Red and Oil Red O 21 days after induction. In Alizarin Red staining, a large number of cells showed mineralization (Fig. 1b). Oil Red O staining revealed numerous intracellular lipid droplets around the nucleus, whereas the cells themselves were long and spindle-shaped (Fig. 1c). In some cases, small lipid droplets merged into larger drops that displaced the nucleus. Lentiviral Transduction of hUC‑MSCs to Express NGF The hUC-MSCs transduced with NGF- and GFP-encoding lentiviruses for 48 h showed green fluorescence (Fig. 2a); the infection rate was > 96%. Stable transformation of the cells was confirmed by the persistence of GFP expression over multiple generations. Given that the same conditions were used for GFP and recombinant NGF lentiviral transduction, these results suggest that NGF was also expressed by these cells. We used non-tranduced hUC-MSCs as a control group. NGF-hUC-MSCs and hUC-MSCs were cultured for 12, 24, and 48 h; NGF levels in the culture supernatant were determined to be 189 ± 13 versus 14 ± 4, 1454 ± 112 versus

48 ± 14 and 4200 ± 331 versus 142 ± 21 pg/ml by ELISA, respectively (Fig. 2c). Neurotrophic Factor Expression in L6–S1 Dorsal Root of Spinal Cord To confirm the capacity of NGF-expressing hUC-MSCs for neural repair, the expression of NGF, BDNF and NT-3 in L6–S1 spinal cord tissue was determined by western blotting and immunohistochemistry after 9 weeks. For semi-quantitative analysis of protein expression, we used the average optical density value (optical density value/area) to reflect the immunohistochemical staining intensity and used the relative grey value (objective band/internal band) to reflect the level of protein expression in western blot. NGF, BDNF, and NT-3 levels were lower in the L6–S1 dorsal root of spinal cord of the diabetic rats than control animals (P < 0.01; Fig. 3 and Supplement 1–2 in Supplementary Material) and were increased in the hUC-MSC and NGF-hUC-MSC groups. In the NGF-hUC-MSC and hUC-MSC transplantation groups, NGF was upregulated relative to the Lentivirus and PBS group (P < 0.01), but its expression was still lower than that in control animals (Fig. 3). The BDNF, NT-3 levels are shown in Supplement 1–2 in Supplementary Material.

Fig.  2  a GFP expression in hUC-MSCs as determined by fluorescence microscopy. b Cells were also visualized by phase-contrast microscopy. c Levels of NGF secreted by hUC-MSCs transduced with an NGF-encoding lentiviral vector and non-transduced hUCMSCs, as determined by ELISA (**P < 0.001)

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Fig. 3  NGF expression in diabetic and control rats with or without hUC-MSC treatment. a Representative images of L6–S1 dorsal root of spinal cord sections (n = 5)from each group. b The mean integral optical density (IOD/area) of positive cells in immunohistochemistry.

c Western blot analysis of NGF protein expression in the L6–S1 dorsal root of spinal cord relative to the level of β-actin. d Mean band intensity ratio for western blot is shown (n = 5 *p < 0.05, **p < 0.01)

Tracking of Transplanted hUC‑MSCs and hUC‑MSC Differentiation in L6–S1 Dorsal Root of Spinal Cord

into neurons and glial cells in the s L6–S1 dorsal root of spinal cord.

We speculated that the repair mechanism not only related to the neurotrophic factor levels observed upon NGF-hUCMSCs injection but also may associated with differentiation of transplanted MSCs. To test this possibility, rats injected with 1 × 106 NGF-hUC-MSCs, MSC, NGF-lentivirus and PBS (n = 5 each) were sacrificed after 42 days for tracking of transplanted hUC-MSCs (Fig. 4) and immunofluorescence analysis. The double-positive yellow cells indicated that the GFP-labeled NGF-hUC-MSCs expressed the neuron-specific protein NeuN or astrocyte marker GFAP, labeled with the white arrow in Fig. 5. The result demonstrated that the engrafted hUC-MSCs differentiated

Cystometry and Histomorphological analysis

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In 9 week, representative cystometric graphs for the rats in each group are shown in Fig. 6a and cystometry statistical data was recorded in Table 1. The micturition interval in each group was 395 ± 36 s for controls, 710 ± 45 for the NGF-hUC-MSC group, 807 ± 54 s for the hUC-MSC group, 1109 ± 76 s for the NGF-LV group and 1277 ± 69 s for the diabetic group. It shows that the micturition interval increase significantly in diabetic rats (P < 0.01), and in NGF-hUC-MSC or hUC-MSC treatment group, it shows a decrease when compared with NGF-LV and PBS group(P < 0.05). The peak point (cm ­H 2O) during

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Fig.  4  a GFP-expressing hUC-MSCs were injected into the L6–S1 spinal cord of diabetic rats; 6  weeks later, many GFP-positive cells were found within the spinal cord tissue relative to that in the spinal

cord tissue of other groups. GFP-positive cells were labeled with the white arrow. b hUC-MSC treatment group (hUC-MSCs). c NGF-lentivirus-only treatment groups (NGF-Lv). d PBS group

Fig. 5  Immunofluorescence was performed to dectect the differentiation of hUC-MSCs. Images are L6–S1 dorsal root of spinal cord sections 6 weeks after transplantation with GFP-labeled hUC-MSCs. GFP-positive cells (green) and specific antibodies against NeuN

(n = 5) and GFAP (n = 5). DAPI staining for cell nuclei (blue). Double-positive cells were labeled with the white arrow. (Color figure online)

each voiding period was 45.7 ± 5.1, 46.4 ± 6.5, 47.9 ± 5.4, 47.6 ± 4.4 and 48.3 ± 5.2 respectively and shows no difference (P > 0.05). Urine volume per void and residual

volume were also examined (Table 1). Hematoxylin and eosin staining revealed a thinner muscle layer with muscle hypertrophy and more fibrous tissue in the bladder of

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Table 1  Comparison of cystometry Groups

Control

NGF-huMSCs

huMSCs

NGF-LV

PBS

Micturition interval (s) Maximum voiding pressure (cm ­H2O) Urine volume per void (mL) Residual volume (mL)

395 ± 36a 45.7 ± 5.1 0.86 ± 0.28a 0.03 ± 0.01a

710 ± 45b 46.4 ± 6.5 2.84 ± 0.34b 0.18 ± 0.05c

807 ± 54b 47.4 ± 5.4 3.41 ± 0.41c 0.24 ± 0.08

1109 ± 76 47.2 ± 4.4 4.08 ± 1.09 0.26 ± 0.11

1277 ± 69 47.3 ± 5.2 4.43 ± 1.12 0.27 ± 0.09

P value < 0.01 > 0.05 < 0.01 < 0.01

a

 P < 0.01 versus other groups

b c

 P < 0.01 versus NGF-LV and PBS.

 P < 0.05 versus NGF-LV and PBS.

Fig.  6  a Cystometric graphs of rats in each group, by time (min) as X-axis and voiding pressure (cm ­H2O) as Y-axis. b Hematoxylin and eosin staining (n = 10 100× for upper panel and 400× for lower panel and black arrows is to show the fibrous tissue)

diabetic rats than was observed in control rats. In addition, the amount of fibrous tissue was significantly reduced in the NGF-hUC-MSC group and hUC-MSC group when compared with NGF-LV and PBS groups (Fig. 6b).

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Discussion Diabetic cystopathy is a complication associated with DM [16] and includes voiding dysfunction, almost every diabetes patients will have this voiding dysfunction in the

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later phase [2, 3]. The effects of NGF on neurons along the micturition pathway in the bladder and spinal cord seem to be farmore complex. And it has become clear that NGF modulates neuronal function along micturition pathways, is involved in multiple bladder pathologies. For example, in the rat model of bladder outlet obstruction (BOO) and detrusor overactivity, the expression of bladder NGF total protein and urinary NGF protein levels were significantly increased [34]. Besides, Yoshimura studied in rat models and demonstrated that intrathecal injection of NGF (2.5 microg/microl) at the L6–S1 level of spinal cord for 1 or 2 weeks could significant increased the level of NGF in L6–S1 dorsal root ganglia and contribute to a time-dependent reduction in intercontraction intervals and hyperexcitability of bladder afferent neurons [35]. Other vitro studies on sensory neurons suggest that NGF may modulate neurotransmitter release, induce synaptic reorganization, increase neuronal excitability by reducing the activation threshold and sensitize nociceptive neurons to noxious stimuli (hypersensitivity) [36, 37]. Contrarily, Seki study investigated the effects of intrathecal application of NGF antibodies (Ab) on bladder hyperreflexia in chronic spinalized rats. In adult female rats, an intrathecal catheter was implanted at the level of the L6 to S1 spinal cord, followed by complete transection of the Th8 to 9 spinal cord. Intrathecal catheter was connected to an osmotic pump for continuous delivery of vehicle or NGF Ab. The results showed that NGF levels in the bladder, L6 spinal cord and L5 to S1 dorsal root ganglia of vehicle treated spinalized rats was 1.6 to 4.8 times higher than in spinal cord intact rats. After intrathecal NGF Ab treatment, NGF levels were significantly lower in the L6 to S1 dorsal root ganglia (30–35%) and L6 spinal cord (53%). Besides, the number of uninhibited bladder contractions per voiding cycle, maximal pressure of uninhibited bladder contraction and maximal voiding pressure were significantly decreased in NGF Ab treated versus vehicle treated spinalized rats [38]. From these studies, it’s not difficult to find that the level of NGF in dorsal root of spinal cord is closely related to bladder function and the relationship between NGF and bladder voiding function is a positive correlation. Furthermore, the voiding function of bladder could be regulated by supplementing or inhibiting the level of NGF by intrathecal injection in L6–S1. In contrast to overactivity of NGF in BOO and SCI, the effection of NGF in diabetic bladder dysfunction. Diabetic cystopathy is a common chronic complication of DM with a variety of lower urinary tract symptoms, hyperglycemia exerts its toxic effects through neuronal impairment. The metabolic derangements lead to axonal degeneration and impairment of nerve conduction, manifesting as bladder hyposensation. The decreased sensation of bladder filling causes bladder overdistention. Repeated bladder

overdistention causes hypocontractility of the diabetic bladder [17]. NGF levels were found to decrease over time in the L6–S1 dorsal root of spinal cord and bladder, leading to bladder voiding dysfunction [12, 15]. Thus, in our study, we refer to their experimental methods in study [35, 38], trying to find a way to improve the level of NGF in the spinal dorsal roots of L6–S1 and improve the voiding symptoms of diabetic bladder cystopathy. Finally, our experimental results also show that this method could be a feasible way in ameliorate diabetic cystopathy in rats. In this study, a rat model of diabetes was generated by STZ administration. Diabetic rats had a lower mean body weight and higher plasma glucose levels than controls, and exhibited voiding dysfunction with the progression of DM, which included increased voiding frequency, urine volume per void, and residual urine volume. However, these symptoms could be prevented and improved upon injection of NGF-hUC-MSCs and hUC-MSCs at an early stage, with the former showing greater effects. Bladder hypertrophy and remodeling also occur in diabetic cystopathy; in the later phase, accumulation of oxidative stress products from prolonged hyperglycemia results in decompensation of bladder tissue and function. We found evidence of decompensation by histomorphological examination of bladder tissue stained with hematoxylin and eosin; for instance, a thinner muscle layer and more fibrous tissue. Surprisingly, we found that the hypertrophy and remodeling were reduced in animals injected with hUC-MSCs, which indicated that early protection of central nervous system of bladder by stem cells could effectively delay the progression of diabetic cystopathy and prevent hypertrophy and remodeling of bladder. In our study, we used hUC-MSCs because they have greater capacity for proliferation than those obtained from bone marrow [10]. Moreover, hUC-MSCs can differentiate into diverse cell types under appropriate conditions and are easy to transfect, with the transfection having no effect on differentiation and proliferation capacities. Studies have shown that decreased levels of NGF are closely associated with disease progression and the exogenous supply of NGF could ameliorate the symptoms. In addition, hUC-MSC transplantation has been reported to be an effective treatment to neurological diseases in rats. In this study, when transfected hUC-MSCs were transplanted into the diabetic rats, not only can they supply exogenous NGF stablely but also can repair the neuropathy by its own cellular function and the results showed a better effect. Besides, the injection method is another problem that should be solved. Previous studies have demonstrated that intrathecal injection of MSCs can potentially be applied to the treatment of central nervous system disease such as: spinal cord injury [22], multiple sclerosis [23], and neurodegenerative diseases [24]; Thus,

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we choose intrathecal injection as the injection method in the present study. It remains unclear how transplanted NGF-hUC-MSCs improve bladder function. As we known NGF, BDNF, and NT-3 are essential neurotrophic factors that regulate neuronal survival and proliferation and control fate specification, axon and dendrite growth, and tissue patterning via Trk receptors [26]. In our study, we demonstrated that hUCMSCs transfected with NGF geen can improve the level of NGF efficiently in L6–S1 dorsal root of spinal cord and the levels of BDNG, NT-3 were also increased. This may related to the transplanted hUC-MSCs’ ability in secreting neurotrophins and cytokines. In conclusion, on one aspect overexpression of NGF through lentiviral transduction can supplement endogenous NGF levels. On the other hand, cytokines and neurotrophin released by transplanted hUCMSCs can stimulate nerve repair and regeneration in diabetic cystopathy. These two ways may explain the possible repair mechanism at molecular and protein levels. Glia are the most abundant cell population in the nervous system. Astrocytes are specialized glia in the central nervous system (CNS) that are essential for its normal functioning; alterations in their activity can contribute to diabetic neuropathy [29]. Reactive astrogliosis encompasses a spectrum of molecular, cellular, and functional changes in astrocytes in response to all forms of CNS injury and disease, including diabetes [28] and increasing gliogenesis can potentially restore damaged neurons. Thus, it is possible that differentiation of transplanted hUC-MSCs into neural cell types underlies the therapeutic effects of hUC-MSCs. Indeed, we found that not only can the hUC-MSCs differentiated into GFAP-positive astrocytes but also differentiated into NeuNpositive neurons when transplanted into the spinal cord. By this way, it helps stabilize the composition of the spinal central nervous system and explains the therapeutic mechanism at a cellular level. In this study, the MSCs transplanted into rats derived from human umbilical cord. Thus, there is a potential risky issue is whether the transplanted MSCs cause immune responses or other adverse reactions in rats. In fact, the application of human derived stem cells in rat models has been widely reported in previous studies[31–33], the treatment effects are quite significant, and serious adverse reactions or complications haven’t been reported yet. But the safety of transplantation of human derived stem cells still need further evidence. In summary, our findings provide evidence that transplantation of hUC-MSCs overexpressing NGF could ameliorate diabetic cystopathy in rats. And it could be a promising therapeutic strategy for diabetic cystopathy in future. Funding  Grant sponsor: National Natural Science Foundation of China; Grant numbers: No. 81170701.

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Neurochem Res Compliance with Ethical Standards  Conflict of interest  There is no conflict of interest regarding the publication of this paper.

References 1. Wild S, Roglic G, Green A, Sicree R, King H (2004) Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 27:1047–1053 2. Frimodt-Møller C (1978) Diabetic cystopathy. A review of the urodynamic and clinical features of neurogenic bladder dysfunction in diabetes mellitus. Dan Med Bull 25:49–60 3. Frimodt-Møller C (1980) Diabetic cystopathy: epidemiology and related disorders. Ann Intern Med 92:318–321 4. Kaplan SA, Blaivas JG (1988) Diabetic cystopathy. J Diabet Complicat 2:133–139 5. Frimodt-Møller C, Mortensen S (1980) Treatment of diabetic cystopathy. Ann Intern Med 92:327–328 6. Huang C, Chu H, Muheremu A, Zuo H (2015) Neurorestorative strategies for Alzheimer’s disease. Neurol India 63:583–588 7. Roper SN, Steindler DA (2013) Stem cells as a potential therapy for epilepsy. Exp Neurol 244:59–66 8. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147 9. Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringdén O (2003) Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 57:11–20 10. Baksh D, Yao R, Tuan RS (2007) Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells 25:1384–1392 11. Fan CG, Zhang QJ, Zhou JR (2011) Therapeutic potentials of mesenchymal stem cells derived from human umbilical cord. Stem Cell Rev 7:195–207 12. Sasaki K, Chancellor MB, Phelan MW, Yokoyama T, Fraser MO, Seki S, Kubo K, Kumon H, Groat WC, Yoshimura N (2002) Diabetic cystopathy correlates with a long-term decrease in nerve growth factor levels in the bladder and lumbosacral dorsal root ganglia. J Urol 168:1259–1264 13. Hellweg R, Raivich G, Hartung HD, Hock C, Kreutzberg GW (1994) Axonal transport of endogenous nerve growth factor (NGF) and NGF receptor in experimental diabetic neuropathy. Exp Neurol 130:24–30 14. Hellweg R, Hartung HD (1990) Endogenous levels of nerve growth factor (NGF) are altered in experimental diabetes mellitus: a possible role of NGF in the pathogenesis of diabetic neuropathy. J Neurosci Res 26:258–267 15. Goins WF, Yoshimura N, Phelan MW, Yokoyama T, Fraser MO, Ozawa H, Bennett N Jr, de Groat WC, Glorioso JC, Chancellor MB (2001) Herpes simplex virus mediated nerve growth factor expression in bladder and afferent neurons: potential treatment for diabetic bladder dysfunction. J Urol 165:1748–1754 16. Sasaki K, Chancellor MB, Goins WF, Phelan MW, Glorioso JC, de Groat WC, Yoshimura N (2004) Gene therapy using replication-defective herpes simplex virus vectors expressing nerve growth factor in a rat model of diabetic cystopathy. Diabetes 53:2723–2730

Neurochem Res 17. Daneshgari F, Liu G, Birder L, Hanna-Mitchell AT, Chacko S (2009) Diabetic bladder dysfunction: current translational knowledge. J Urol 182:S18–S26 18. Koh SH, Kim KS, Choi MR, Jung KH, Park KS, Chai YG, Roh W, Hwang SJ, Ko HJ, Huh YM, Kim HT, Kim SH (2008) Implantation of human umbilical cord-derived mesenchymal stem cells as a neuroprotective therapy for ischemic stroke in rats. Brain Res 1229:233–248 19. Liao W, Xie J, Zhong J, Liu Y, Du L, Zhou B, Xu J, Liu P, Yang S, Wang J, Han Z, Han ZC (2009) Therapeutic effect of human umbilical cord multipotent mesenchymal stromal cells in a rat model of stroke. Transplantation 87:350–359 20. Fu YS, Cheng YC, Lin MY, Cheng H, Chu PM, Chou SC, Shih YH, Ko MH, Sung MS (2006) Conversion of human umbilical cord mesenchymal stem cells in Wharton’s jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism. Stem Cells 24:115–124 21. Yang CC, Shih YH, Ko MH, Hsu SY, Cheng H, Fu YS (2008) Transplantation of human umbilical mesenchymal stem cells from Wharton’s jelly after complete transection of the rat spinal cord. PLoS ONE 3:e3336 22. Kishk NA, Gabr H, Hamdy S, Afifi L, Abokresha N, Mahmoud H, Wafaie A, Bilal D (2010) Case control series of intrathecal autologous bone marrow mesenchymal stem cell therapy for chronic spinal cord injury. Neurorehabil Neural Repair 24:702–708 23. Karussis D, Karageorgiou C, Vaknin-Dembinsky A, GowdaKurkalli B, Gomori JM, Kassis I, Bulte JW, Petrou P, Ben-Hur T, Abramsky O, Slavin S (2010) Safety and immunological effects of mesenchymal stem cell transplantation in patients with multiple sclerosis and amyotrophic lateral sclerosis. Arch Neurol 67:1187–1194 24. Joyce N, Annett G, Wirthlin L, Olson S, Bauer G, Nolta JA (2010) Mesenchymal stem cells for the treatment of neurodegenerative disease. Regen Med 5:933–946 25. Lim JY, Jeong CH, Jun JA, Kim SM, Ryu CH, Hou Y, Oh W, Chang JW, Jeun SS (2011) Therapeutic effects of human umbilical cord blood-derived mesenchymal stem cells after intrathecal administration by lumbar puncture in a rat model of cerebral ischemia. Stem Cell Res Ther 2:38 26. Huang EJ, Reichardt LF (2003) Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem 72:609–642

27. Sarugaser R, Lickorish D, Baksh D, Hosseini MM, Davies JE (2005) Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells 23:220–229 28. Takahashi S, Izawa Y, Suzuki N (2012) Astroglial pentose phosphate pathway rates in response to high-glucose environments. ASN Neuro 4:e00078 29. Coleman E, Judd R, Hoe L, Dennis J, Posner P (2004) Effects of diabetes mellitus on astrocyte GFAP and glutamate transporters in the CNS. Glia 48:166–178 30. Huang YC, Shindel AW, Ning H, Lin G, Harraz AM, Wang G, Garcia M, Lue TF, Lin CS (2010) Adipose derived stem cells ameliorate hyperlipidemia associated detrusor overactivity in a rat model. J Urol 183:1232–1240 31. Hsueh Yuan-Yu, Chang Ya-Ju, Huang Chia-Wei et  al (2016) Synergy of endothelial and neural progenitor cells from adiposederived stem cells to preserve neurovascular structures in rat hypoxic-ischemic brain injury. Sci Rep 5:14985 32. Huang C, Zhao L, Gu J, Nie D, Chen Y, Zuo H, Huan W, Shi J, Chen J, Shi W (2016) The migration and differentiation of hUCMSCsCXCR4/GFP encapsulated in BDNF/chitosan scaffolds for brain tissue engineering. Biomed Mater 11(3):035004 33. Chen C, Chen F, Yao C, Shu S, Feng J, Hu X, Hai Q, Yao S, Chen X (2016) Intrathecal injection of human umbilical cord-derived mesenchymal stem cells ameliorates neuropathic pain in rats. Neurochem Res. 41(12):3250–3260 34. Lee SR, Hong CH, Choi YD et al (2010) Increased urinary nerve growth factor as apredictor of persistent detrusor overactivity after bladder outlet obstruc-tion relief in a rat model. J Urol 183:2440–2444 35. Yoshimura N, Bennett NE, Hayashi Y et al (2006) Bladder overactivity and hyperexcitability of bladder afferent neurons after intrathecal delivery of nerve growth factor in rats. J Neurosci 26(42):10847–10855 36. Hu VY, Zvara P, Dattilio A et al (2005) Decrease in bladder overactivity with REN1820 in rats with cyclophosphamide induced cystitis. J Urol 173:1016–1021 37. Pezet S, McMahon SB. Neurotrophins (2006) Mediators and modulators of pain. Annu Rev Neurosci 29:507–538 38. Seki S, Sasaki K, Fraser MO et al (2002) Immunoneutralization of nerve growth factor in lumbosacral spinal cord reduces bladder hyperreflexia in spinal cord injured rats. J Urol 168(5):2269–2274

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