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Lowering the concentration affects the migration and viability of intracerebroventricular-delivered human mesenchymal stem cells
Biochemical and Biophysical Research Communications xxx (2017) 1e7
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Lowering the concentration affects the migration and viability of intracerebroventricular-delivered human mesenchymal stem cells Hyeong Seop Kim a, b, c, d, 1, Na Kyung Lee a, b, c, d, 1, Dongkyeom Yoo e, Jeongmin Lee a, b, c, d, Soo Jin Choi f, Wonil Oh f, Jong Wook Chang a, d, **, Duk L. Na a, b, c, d, * a
Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, 06351, Republic of Korea Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 06351, Republic of Korea Neuroscience Center, Samsung Medical Center, Seoul, 06351, Republic of Korea d Stem Cell & Regenerative Medicine Institute, Samsung Medical Center, Seoul, 06351, Republic of Korea e Center for Molecular & Cellular Imaging, Samsung Biomedical Research Institute, Seoul, 06351, Republic of Korea f Biomedical Research Institute, MEDIPOST Co., Ltd., 463-400, Gyeonggi-do, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 August 2017 Accepted 27 August 2017 Available online xxx
Due to their widely known therapeutic benefits, mesenchymal stem cells have been proposed as a novel treatment option for a wide range of diseases including Alzheimer's disease. To maximize these benefits, critical factors such as delivery route, cell viability, and cell migration must be accounted for. Out of the various delivery routes to the brain, the intracerebroventricular (ICV) route stands out due to the widespread distribution that can occur via cerebrospinal fluid flow. The major objective of this present study was to observe how altering cell concentration influences the migration and viability of human umbilical cord blood derived-mesenchymal stem cells (hUCB-MSCs), delivered via ICV injection, in the brains of wild-type (WT) mice. C3H/C57 WT mice were divided into three groups and were injected with 1 105 hUCB-MSCs suspended in varying volumes: high (3 ml), middle (5 ml), and low (7 ml) concentrations, respectively. Lowering the concentration increased the migratory capabilities and elevated the viability of hUCB-MSCs. These results suggest that cell concentration can affect the physiological state of hUCB-MSCs, and thus the extent of therapeutic efficacy that can be achieved. © 2017 Published by Elsevier Inc.
Keywords: Human mesenchymal stem cell Distribution Concentration Migration Viability
1. Introduction Mesenchymal stem cells (MSCs) have risen as a highly attractive candidate for stem cell therapy due to their multiple functional
Abbreviations: ICV, intracerebroventricular; CSF, cerebrospinal fluid; hUCB-MSC, human umbilical cord blood derived-mesenchymal stem cell; IHC, immunohistochemical; PB, prussian blue; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; AD, Alzheimer's disease; BBB, blood-brain barrier; ALU, arthrobacter luteus; PBMC, peripheral blood mononuclear cell. * Corresponding author. Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea. ** Corresponding author. Stem Cell & Regenerative Medicine Institute, Samsung Medical Center, Seoul, Republic of Korea, Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, 81 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea. E-mail addresses: [email protected] (J.W. Chang), dukna@naver. com (D.L. Na). 1 Both authors contributed equally to this work.
roles and their mechanism of action is thought to be primarily mediated via paracrine activity which is the secretion of various proteins into the external environment [1e3]. Through such an action, MSCs not only aid in reducing the pathological cause of a disease but also contribute towards tissue regeneration [4,5]. In order for MSCs to exert their therapeutic action at the target site, such as the brain in this study, the delivery route is an essential factor that must be considered. Based on our pre-clinical and clinical studies centered on Alzheimer's disease (AD), intraparenchymal and intracerebroventricular (ICV) administration seem to be the potential delivery routes for stem cell therapy [6]. Each route has its pros and cons; for instance, direct transplantation to the target site is possible through the intra-parenchymal administration but it is invasive and temporary mechanical injury results following insertion of the needle. On the other hand, widespread distribution of the therapeutic agent is possible through the ICV route by utilizing the cerebrospinal fluid (CSF) flow [7]. Moreover, the therapeutic effects that arise from ICV injection of MSCs have also been
http://dx.doi.org/10.1016/j.bbrc.2017.08.115 0006-291X/© 2017 Published by Elsevier Inc.
Please cite this article in press as: H.S. Kim, et al., Lowering the concentration affects the migration and viability of intracerebroventriculardelivered human mesenchymal stem cells, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.115
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proposed by various groups [8,9]. Although considered a suitable administration route to deliver MSCs into the brain, few studies have endeavored upon adjusting factors such as cell concentration and volume, prior to transplantation, so as to maximize the therapeutic benefits that can be gained from the injected cells. Past groups have used different cell concentrations when performing ICV injections in mouse models [9e11]. Based on a preliminary study, we noted that ICV-injected MSCs showed a tendency to aggregate into a spherical mass inside the lateral ventricle. This brought to question how concentration can affect the physiological state of MSCs. With this in mind, the major objective of this study was to investigate on how altering cell concentration affects the migration and viability of human umbilical cord blood derived-mesenchymal stem cells (hUCBMSCs) following delivery into the wild-type (WT) mouse brain via the ICV route.
were injected at a rate of 0.5 ml per min by using a 25 ml Hamilton syringe (Hamilton Company, Reno, Nevada, USA). The syringe was removed after a 5-min delay to prevent the loss of cells through the needle track.
2. Materials and methods
2.6. Tissue preparation and histological analysis
2.1. Ethical statement
At post 1 day, mice were sacrificed through cardiac perfusion with PBS. The harvested brain tissue samples were fixed in 4% paraformaldehyde (PFA; Biosesang, Republic of Korea) for 24 h before being embedded in paraffin. 4 mm-thick-coronal sections were obtained using a microtome (Leica Biosystems, Wetzlar, Germany). By referring to a previously reported protocol [15], immunostaining was conducted by using the following primary: antihuman mitochondria (1:200; Merck Millipore, MS, USA) and secondary: biotin-conjugated anti-mouse (1:2000; Vector Laboratories, Inc., CA, USA) antibodies. Reactivity was visualized using the 3,30 -Diaminobenzidine (DAB) solution (Vector Laboratories, Inc.) and counter staining was performed by hematoxylin (Dako, CA, USA). Slides were analyzed by a Scanscope AT scanner (Leica Biosystems). Prussian blue staining was done as instructed by the manufacturer (IHC World, CA, USA). Slides were treated with equal volumes of potassium ferricyanide and hydrochloric acid for 30 min and were then washed with distilled water (D.W.). Nuclear fast red was used for counterstaining and slides were washed with D.W. afterwards. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (in situ Cell Death Detection Kit, Roche Applied Science, IN, USA) was performed according to the manufacturer's instructions. Images were acquired using a confocal microscope (Carl Zeiss AG, Jena, Germany). The average percentage of TUNEL-positive cells was quantified from at least 3 random fields for each experimental group.
This study has been approved by the Institutional Animal Care and Use Committee (IACUC) of the Samsung Biomedical Research Institute (SBRI) at Samsung Medical Center (SMC). As an accredited facility of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International), SBRI acts in accordance to the guidelines sets by the Institute of Laboratory Animal Resources (ILAR). Approval was received from the institutional ethical review board (IRB) of SMC to collect cerebrospinal fluid (CSF) samples from human subjects (IRB 2015-04-099019). Written informed consent was obtained from all subjects. CSF samples were collected and stored according to a procedure reported previously [12]. 2.2. Preparation of ferumoxytol-labeled hUCB-MSCs Human umbilical cord blood-derived mesenchymal stem cells (MEDIPOST Inc., Biomedical Research Institute Co., Ltd, Republic of Korea) were labeled with ferumoxytol (Rienso®; Takeda Inc., UK) using previously reported methods [13,14]. On the day of the surgical procedure, 1 105 of the ferumoxytol-labeled hUCB-MSCs were suspended in varying volumes of phenol red free MEMa1x media (Gibco-Invitrogen): 3, 5, and 7 ml for the high, middle, and low concentration groups, respectively.
2.5. MR monitoring following ICV injection Pre and post 1 day MR images were taken using a 7T small animal MR system (Bruker-Biospin, F€ allanden, Switzerland) equipped with a Paravision software (version 6.0). As described previously, the following parameters: (repetition time) TR/(echo time) TE ¼ 2500/20 msec, slice thickness ¼ 0.7 mm, and number of averages ¼ 8, were used to acquire T2-weighted MR images; TR/ TE ¼ 303.805/18 msec, slice thickness ¼ 0.7 mm, flip angle (FA) ¼ 45 , and number of averages ¼ 8, were used to obtain T2* gradient echo MR images [15,16].
2.3. Experimental animals 2.7. Real-time quantitative PCR C3H/C57 (crossbred from C57BL/6 and C3H/HeJ mice) mice that were 7e10 months old were used for this study. Each mouse strain was purchased from the Jackson Laboratory (USA). A total of twenty-one mice were used in this study: MRI and histology: n ¼ 3 (high), n ¼ 3 (middle), and n ¼ 3 (low); quantitative real-time PCR: n ¼ 3 (control; vehicle), n ¼ 3 (high), n ¼ 3 (middle), and n ¼ 3 (low). 2.4. Intracerebroventricular injection of ferumoxytol-labeled hUCBMSCs Before surgery, all of the experimental animals were initially anesthetized by 5% isoflurane. During the surgical procedure, anesthesia was maintained at 2% isoflurane, the mice were fastened using a rodent universal surgical stereotactic frame (Harvard apparatus, Holliston, MA, USA), and ferumoxytol-labeled hUCBMSCs were injected into the right lateral ventricle at the following coordinates: A/P: 0.4 mm, M/L: þ1.0 mm, and D/V: 2.3 mm. Cells
After being sacrificed through cardiac perfusion, coronal sections of the harvested brain tissues from each mouse were sliced in 2 mm thickness by using a brain matrix. Tissues were frozen in liquid nitrogen and then ground up. Genomic DNA extraction (QIAGEN, Hilden, Germany) was performed from tissue samples that weighed around 20 mg. Real time polymerase chain reaction (real-time PCR; QuantStudio, Life Technologies, USA) was carried out by analyzing 10 ng of genomic DNA per sample by using the SYBR Green Master Mix probe (Thermo Fisher Scientific, MA, USA) and primers that targeted the Arthrobacter luteus (ALU) gene. The following primers were used: 50 -GTC AGG AGA TCG AGA CCA TCC C30 (ALU; forward) and 50 -TCC TGC CTC AGC CTC CCA AG-30 (ALU; reverse). A total of 40 cycles were run to amplify the 20 ml PCR reactions and the steps for each stage were as follows: initial hold (95 C, 10 min), denaturation (95 C, 15 s), annealing (68 C, 30 s), and extension (72 C, 30 s). The amount of DNA or number of hUCBMSCs, was calculated by fitting the respective threshold cycle (CT)
Please cite this article in press as: H.S. Kim, et al., Lowering the concentration affects the migration and viability of intracerebroventriculardelivered human mesenchymal stem cells, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.115
Fig. 1. MR images of ICV-injected ferumoxytol-labeled hUCB-MSCs with varying concentrations. T2 weighted and T2* MR images reveal the presence of hypo-intense signals (indicated by red arrows) in the lateral ventricles for all three concentration groups (A) high, (B) middle, and (C) low. The higher the concentration, the MR signal seemed more pronounced. Signals were also detected in the contralateral side for the low concentration group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Histological confirmation of MR images. Prussian blue and immunohistochemical (IHC) stains of sections corresponding to the MR images for the (A) high, (B) middle, and (C) low concentration groups. The blue (prussian blue) and human mitochondria (IHC) stains confirmed the presence of ferumoxytol-labeled hUCB-MSCs at the site of hypointensity. Cellular distribution is greater with decrease in cell concentration. Prussian blue: scale bar ¼ 1 mm (left), 100 mm (right), IHC: scale bar ¼ 100 mm.
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known to have an important role in migration [17], total RNA was isolated from the samples using TRIzol (Invitrogen, MA, USA). cDNA was synthesized from 1000 ng of RNA from each of the samples by following the manufacturer's instructions (Superscript III Reverse Transcriptase; Invitrogen, USA). PCR cycling conditions (40 cycles) were carried out as follows: initial hold (95 C, 10 min), denaturation (95 C, 15 s), annealing (60 C, 1 min), to amplify the 20 ml reaction mixtures. Analysis of relative gene expression data was carried out by using the comparative CT method [18]. 2.8. CCK8 and LDH assay The in vivo experiment was recapitulated in vitro where the equivalent number and concentration of cells for each group was injected into ultra-low attachment 96 well plate (Costar, NY, USA) wells (in triplicates) containing 100 ml of CSF at the equivalent rate of 0.5 ml per min by using the same stereotactic system used for the animal experiment. After incubation in 37 C, 5% CO2 for a day, cell viability was measured using the cell counting kit-8 (CCK8, Dojindo Laboratories, Japan). Lactate dehydrogenase activity was evaluated based on previously reported procedures [19,20]. The fluorescence of both of the assays was read using a microplate spectrophotometer (Bio-rad, CA, USA). CSF only wells (in triplicates) were prepared for background subtraction for both assays. 2.9. Statistical analysis All values are represented as mean ± standard error of mean (S.E.M). One-way ANOVA (Tukey post-hoc) and paired t tests were used to assess significance and a p-value 0.05 was considered statistically significant (GraphPad Software, CA, USA). 3. Results 3.1. Effects of cell concentration on distribution of ferumoxytollabeled hUCB-MSCs based on MRI
Fig. 3. Quantitative assessment of the anatomical distributions of hUCB-MSCs in the mouse brain. (A) Schematic illustration showing the four different 2 mm coronal brain sections (regions 1, 2, 3, 4) that were acquired and analyzed for real-time PCR. (B) Plotted are the average numbers of MSCs present within each of the respective brain sections for the following groups: control, high, middle, and low. Compared to the high concentration group, a significant difference existed in the average number of engrafted MSCs for the middle and low concentration groups. (C) Total number (average) of residual cells remaining in the brains (sum of the four regions) of mice from each of the groups. Decreasing the concentration resulted in a greater loss of cells. All data are shown as mean ± S.E.M.; *p < 0.05 vs. the high group.
values to a standard curve that was created by varying the number of hUCB-MSCs (102, 103, 104, 105, and 106) mixed with mouse peripheral blood mononuclear cells (PBMCs) to make a total of 1 106 cells. To observe changes in mRNA expression of the C-X-C chemokine receptor type 4 (CXCR4) gene (forward: 50 -GAG TCG ATG CTG ATC CCA AT-30 , reverse: 50 -AAG GCT ATC AGA AGC GCA AG-30 ), a gene
For each of the groups, ferumoxytol-labeled hUCB-MSCs appeared as dark, hypo-intense signals in the T2 weighted and T2* post MR images (Fig. 1). With increase in cell concentration, the signal appeared focal and more prominent at the site of injection. For example, a dark aggregated mass (red arrows), suggesting the transplanted cell aggregate, was observed in the right lateral ventricle of the high concentration group (Fig. 1A). The size of the predicted transplanted aggregate reduced with decrease in cell concentration according to the T2 MR images (Fig. 1B and C). For the low concentration group, hypo-intense signals were not only detected at the site of transplantation, but also at various regions of the brain including the contralateral side, which indicated a possible increase in cellular distribution following reduction in concentration (Fig. 1C). 3.2. Histological assessment of cellular distribution At the sites where hypo-intense signals were visualized, human MSCs were detected by using the human-specific anti-mitochondria antibody (Fig. 2). The histological stains confirmed that the source of the MR signals was from the transplanted ferumoxytollabeled hUCB-MSCs. Different paraffin sections from each group were selected to identify the localization of MSCs (Fig. 2A). Similar to previous reports, the transplanted MSCs were stained as dark brown, small specks that surrounded the nucleus [15,16]. Although faint, iron positive, blue signals were also observed at the site of MSC engraftment through prussian blue staining. According to the histological stains, the size of the transplanted aggregate cell mass
Please cite this article in press as: H.S. Kim, et al., Lowering the concentration affects the migration and viability of intracerebroventriculardelivered human mesenchymal stem cells, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.115
H.S. Kim et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
was larger near the injection site for the high compared to the middle concentration group (Fig. 2B). Cell aggregates were detected at regions far from the injection site for the low concentration group, which provided further evidence that cell distribution increases with decrease in cell concentration (Fig. 2C). 3.3. Quantitative evaluation on the effects of concentration in cell distribution To quantify the effects of concentration in the overall distribution of ferumoxytol-labeled hUCB-MSCs, the amounts of humanspecific DNA sequences in the mouse brains were quantified by real-time PCR. Four different coronal sections were obtained from each mouse according to Fig. 3A. The higher the concentration, the
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number of human cells significantly increased at the site of injection, region 2 (Fig. 3B). The total number of MSCs was 2.2 and 4.9folds greater when comparing high vs. middle and low concentrations, respectively. Although not as striking as the high group, a greatest number of MSCs was also observed in region 2 for the middle group. For the low concentration group, however, the number of human MSCs varied among the coronal brain sections. Interestingly, unlike the high and middle groups, the greatest number of human cells was detected in region 1 for the low group (Fig. 3B). When assessing the total number of cells present for each group, even though a more widespread distribution of the cells was achieved, decreasing the concentration attributed to an overall loss of cell number (Fig. 3C).
Fig. 4. Assessment of the effects of concentration on cell viability and migration. (A) Confocal images (scale bar ¼ 20 mm) and (B) quantitative analysis of TUNEL-positive cells for each group. TUNEL assay was performed using adjacent sections remaining after histological staining. The confocal images represent higher magnifications of the DAB images shown in box insets on the left. The yellow, broken line indicates the location of the MSC aggregate. Results are represented as mean ± S.E.M. of >3 random fields per group; **p < 0.01. In vitro evaluation of cell death carried out by using the (C) CCK8 and (D) LDH assays. Results are represented as mean ± S.E.M. of three independent experiments; *p < 0.05. (D) Relative gene expression levels of CXCR4. Results are represented as mean ± S.E.M. of three independent experiments; ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: H.S. Kim, et al., Lowering the concentration affects the migration and viability of intracerebroventriculardelivered human mesenchymal stem cells, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.115
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3.4. Assessment of differences in cell viability and migration
Author contribution
Since the overall trend in cellular distribution has been confirmed, for the experiments carried out from here on, differences in cell viability and migration were compared between the high and low concentrations. To evaluate the viability of transplanted MSCs in vivo, adjacent sections remaining after immunohistochemical staining were selected to perform the TUNEL assay (Fig. 4A). Overall, the percentage of TUNEL-positive cells was below 20%, but a striking and statistically significant difference was noted between the high and low concentration groups (Fig. 4B). An in vitro study was also additionally performed to recapitulate the in vivo results. MSCs were injected into 96-well plate wells containing CSF to mimic the injection of cells into the lateral ventricle in vivo. Compared to the TUNEL assay, similar trends were observed from the CCK8 (Fig. 4C) and LDH (Fig. 4D) assays. The low concentration group showed the highest viability (1.87-fold increase compared to the high concentration group) and lowest LDH release (1.38-fold increase in LDH release was evident for the high concentration group), respectively. Interestingly, CXCR4 expression was 1.5-fold higher for the low concentration group, thus highlighting enhanced migratory abilities with decrease in cell concentration (Fig. 4E).
HSK and NKL equally performed the overall experiments. HSK, NKL, JWC, and DLN designed and conceived the study. DKY acquired the MR images. JML collected and stored the CSF samples. SJC and WIO received permission to use the hUCB-MSCs. HSK, NKL, JWC, and DLN wrote and revised the manuscript. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment We would like to thank Jong Sin Park for providing help with the graphical illustrations. This study was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C3484), the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C2746), and Basic Research Program through the National Research Foundation of South Korea (NRF) funded by the Ministry of Education (NRF-2014R1A2A1A11050576). Transparency document
4. Discussion Other than widespread distribution, ICV administration has low surgical trauma and MSCs delivered through this route are also capable of activating stem cells residing in the subventricular zone [21e23]. The barrier between the brain parenchyma and ventricle is more permeable than that of the blood brain barrier (BBB), thus increasing the changes of MSC penetration into the brain parenchyma [24]. In one of our recent studies, we have observed the migration of MSCs into the brain parenchyma of a canine model when administered via the ICV route. Based on review of prior studies, varying concentrations such as 2.5 105 cells in 2 ml or 5 104 cells in 1 ml (both are higher than the concentration of the “high” group in this study) were used when performing ICV injections [9e11]. Although the feasibility of the ICV route in delivering various concentrations of cells have been diversely reported, how varying cell concentration affects the overall physiological state of MSCs in terms of migration and viability have not been closely investigated. According to the results of this study, MSCs were observed at the site of injection as well as various regions of the brain for all three concentrations (high, middle, and low). The lower the concentration, a more widespread distribution of the MSCs was noted. Contrastingly, MSCs delivered with a high concentration showed higher localization near the site of injection. The low concentration group also showed the highest viability. Such results are indicative that MSCs injected at low concentrations are more capable of exerting therapeutic action through secretion of paracrine factors in comparison to cells injected at higher concentrations. Paracrine factors secreted by MSCs, however, may encounter dilution effects from the surrounding CSF. Additionally, loss in total number of cells is highest for the low concentration group. Thus, although widespread migration can be achieved, when injected at low concentrations, the overall number of remaining cells after washout will be too low to generate therapeutic benefits. This loss, however, can be compensated by performing multiple injections. For future studies, considering such factors will serve beneficial roles when applied to numerous diseases such as AD where pathology is not localized.
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Lowering the concentration affects the migration and viability of intracerebroventricular-delivered human mesenchymal stem cells Hyeong Seop Kim a, b, c, d, 1, Na Kyung Lee a, b, c, d, 1, Dongkyeom Yoo e, Jeongmin Lee a, b, c, d, Soo Jin Choi f, Wonil Oh f, Jong Wook Chang a, d, **, Duk L. Na a, b, c, d, * a
Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, 06351, Republic of Korea Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 06351, Republic of Korea Neuroscience Center, Samsung Medical Center, Seoul, 06351, Republic of Korea d Stem Cell & Regenerative Medicine Institute, Samsung Medical Center, Seoul, 06351, Republic of Korea e Center for Molecular & Cellular Imaging, Samsung Biomedical Research Institute, Seoul, 06351, Republic of Korea f Biomedical Research Institute, MEDIPOST Co., Ltd., 463-400, Gyeonggi-do, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 August 2017 Accepted 27 August 2017 Available online xxx
Due to their widely known therapeutic benefits, mesenchymal stem cells have been proposed as a novel treatment option for a wide range of diseases including Alzheimer's disease. To maximize these benefits, critical factors such as delivery route, cell viability, and cell migration must be accounted for. Out of the various delivery routes to the brain, the intracerebroventricular (ICV) route stands out due to the widespread distribution that can occur via cerebrospinal fluid flow. The major objective of this present study was to observe how altering cell concentration influences the migration and viability of human umbilical cord blood derived-mesenchymal stem cells (hUCB-MSCs), delivered via ICV injection, in the brains of wild-type (WT) mice. C3H/C57 WT mice were divided into three groups and were injected with 1 105 hUCB-MSCs suspended in varying volumes: high (3 ml), middle (5 ml), and low (7 ml) concentrations, respectively. Lowering the concentration increased the migratory capabilities and elevated the viability of hUCB-MSCs. These results suggest that cell concentration can affect the physiological state of hUCB-MSCs, and thus the extent of therapeutic efficacy that can be achieved. © 2017 Published by Elsevier Inc.
Keywords: Human mesenchymal stem cell Distribution Concentration Migration Viability
1. Introduction Mesenchymal stem cells (MSCs) have risen as a highly attractive candidate for stem cell therapy due to their multiple functional
Abbreviations: ICV, intracerebroventricular; CSF, cerebrospinal fluid; hUCB-MSC, human umbilical cord blood derived-mesenchymal stem cell; IHC, immunohistochemical; PB, prussian blue; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; AD, Alzheimer's disease; BBB, blood-brain barrier; ALU, arthrobacter luteus; PBMC, peripheral blood mononuclear cell. * Corresponding author. Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea. ** Corresponding author. Stem Cell & Regenerative Medicine Institute, Samsung Medical Center, Seoul, Republic of Korea, Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, 81 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea. E-mail addresses: [email protected] (J.W. Chang), dukna@naver. com (D.L. Na). 1 Both authors contributed equally to this work.
roles and their mechanism of action is thought to be primarily mediated via paracrine activity which is the secretion of various proteins into the external environment [1e3]. Through such an action, MSCs not only aid in reducing the pathological cause of a disease but also contribute towards tissue regeneration [4,5]. In order for MSCs to exert their therapeutic action at the target site, such as the brain in this study, the delivery route is an essential factor that must be considered. Based on our pre-clinical and clinical studies centered on Alzheimer's disease (AD), intraparenchymal and intracerebroventricular (ICV) administration seem to be the potential delivery routes for stem cell therapy [6]. Each route has its pros and cons; for instance, direct transplantation to the target site is possible through the intra-parenchymal administration but it is invasive and temporary mechanical injury results following insertion of the needle. On the other hand, widespread distribution of the therapeutic agent is possible through the ICV route by utilizing the cerebrospinal fluid (CSF) flow [7]. Moreover, the therapeutic effects that arise from ICV injection of MSCs have also been
http://dx.doi.org/10.1016/j.bbrc.2017.08.115 0006-291X/© 2017 Published by Elsevier Inc.
Please cite this article in press as: H.S. Kim, et al., Lowering the concentration affects the migration and viability of intracerebroventriculardelivered human mesenchymal stem cells, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.115
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proposed by various groups [8,9]. Although considered a suitable administration route to deliver MSCs into the brain, few studies have endeavored upon adjusting factors such as cell concentration and volume, prior to transplantation, so as to maximize the therapeutic benefits that can be gained from the injected cells. Past groups have used different cell concentrations when performing ICV injections in mouse models [9e11]. Based on a preliminary study, we noted that ICV-injected MSCs showed a tendency to aggregate into a spherical mass inside the lateral ventricle. This brought to question how concentration can affect the physiological state of MSCs. With this in mind, the major objective of this study was to investigate on how altering cell concentration affects the migration and viability of human umbilical cord blood derived-mesenchymal stem cells (hUCBMSCs) following delivery into the wild-type (WT) mouse brain via the ICV route.
were injected at a rate of 0.5 ml per min by using a 25 ml Hamilton syringe (Hamilton Company, Reno, Nevada, USA). The syringe was removed after a 5-min delay to prevent the loss of cells through the needle track.
2. Materials and methods
2.6. Tissue preparation and histological analysis
2.1. Ethical statement
At post 1 day, mice were sacrificed through cardiac perfusion with PBS. The harvested brain tissue samples were fixed in 4% paraformaldehyde (PFA; Biosesang, Republic of Korea) for 24 h before being embedded in paraffin. 4 mm-thick-coronal sections were obtained using a microtome (Leica Biosystems, Wetzlar, Germany). By referring to a previously reported protocol [15], immunostaining was conducted by using the following primary: antihuman mitochondria (1:200; Merck Millipore, MS, USA) and secondary: biotin-conjugated anti-mouse (1:2000; Vector Laboratories, Inc., CA, USA) antibodies. Reactivity was visualized using the 3,30 -Diaminobenzidine (DAB) solution (Vector Laboratories, Inc.) and counter staining was performed by hematoxylin (Dako, CA, USA). Slides were analyzed by a Scanscope AT scanner (Leica Biosystems). Prussian blue staining was done as instructed by the manufacturer (IHC World, CA, USA). Slides were treated with equal volumes of potassium ferricyanide and hydrochloric acid for 30 min and were then washed with distilled water (D.W.). Nuclear fast red was used for counterstaining and slides were washed with D.W. afterwards. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (in situ Cell Death Detection Kit, Roche Applied Science, IN, USA) was performed according to the manufacturer's instructions. Images were acquired using a confocal microscope (Carl Zeiss AG, Jena, Germany). The average percentage of TUNEL-positive cells was quantified from at least 3 random fields for each experimental group.
This study has been approved by the Institutional Animal Care and Use Committee (IACUC) of the Samsung Biomedical Research Institute (SBRI) at Samsung Medical Center (SMC). As an accredited facility of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International), SBRI acts in accordance to the guidelines sets by the Institute of Laboratory Animal Resources (ILAR). Approval was received from the institutional ethical review board (IRB) of SMC to collect cerebrospinal fluid (CSF) samples from human subjects (IRB 2015-04-099019). Written informed consent was obtained from all subjects. CSF samples were collected and stored according to a procedure reported previously [12]. 2.2. Preparation of ferumoxytol-labeled hUCB-MSCs Human umbilical cord blood-derived mesenchymal stem cells (MEDIPOST Inc., Biomedical Research Institute Co., Ltd, Republic of Korea) were labeled with ferumoxytol (Rienso®; Takeda Inc., UK) using previously reported methods [13,14]. On the day of the surgical procedure, 1 105 of the ferumoxytol-labeled hUCB-MSCs were suspended in varying volumes of phenol red free MEMa1x media (Gibco-Invitrogen): 3, 5, and 7 ml for the high, middle, and low concentration groups, respectively.
2.5. MR monitoring following ICV injection Pre and post 1 day MR images were taken using a 7T small animal MR system (Bruker-Biospin, F€ allanden, Switzerland) equipped with a Paravision software (version 6.0). As described previously, the following parameters: (repetition time) TR/(echo time) TE ¼ 2500/20 msec, slice thickness ¼ 0.7 mm, and number of averages ¼ 8, were used to acquire T2-weighted MR images; TR/ TE ¼ 303.805/18 msec, slice thickness ¼ 0.7 mm, flip angle (FA) ¼ 45 , and number of averages ¼ 8, were used to obtain T2* gradient echo MR images [15,16].
2.3. Experimental animals 2.7. Real-time quantitative PCR C3H/C57 (crossbred from C57BL/6 and C3H/HeJ mice) mice that were 7e10 months old were used for this study. Each mouse strain was purchased from the Jackson Laboratory (USA). A total of twenty-one mice were used in this study: MRI and histology: n ¼ 3 (high), n ¼ 3 (middle), and n ¼ 3 (low); quantitative real-time PCR: n ¼ 3 (control; vehicle), n ¼ 3 (high), n ¼ 3 (middle), and n ¼ 3 (low). 2.4. Intracerebroventricular injection of ferumoxytol-labeled hUCBMSCs Before surgery, all of the experimental animals were initially anesthetized by 5% isoflurane. During the surgical procedure, anesthesia was maintained at 2% isoflurane, the mice were fastened using a rodent universal surgical stereotactic frame (Harvard apparatus, Holliston, MA, USA), and ferumoxytol-labeled hUCBMSCs were injected into the right lateral ventricle at the following coordinates: A/P: 0.4 mm, M/L: þ1.0 mm, and D/V: 2.3 mm. Cells
After being sacrificed through cardiac perfusion, coronal sections of the harvested brain tissues from each mouse were sliced in 2 mm thickness by using a brain matrix. Tissues were frozen in liquid nitrogen and then ground up. Genomic DNA extraction (QIAGEN, Hilden, Germany) was performed from tissue samples that weighed around 20 mg. Real time polymerase chain reaction (real-time PCR; QuantStudio, Life Technologies, USA) was carried out by analyzing 10 ng of genomic DNA per sample by using the SYBR Green Master Mix probe (Thermo Fisher Scientific, MA, USA) and primers that targeted the Arthrobacter luteus (ALU) gene. The following primers were used: 50 -GTC AGG AGA TCG AGA CCA TCC C30 (ALU; forward) and 50 -TCC TGC CTC AGC CTC CCA AG-30 (ALU; reverse). A total of 40 cycles were run to amplify the 20 ml PCR reactions and the steps for each stage were as follows: initial hold (95 C, 10 min), denaturation (95 C, 15 s), annealing (68 C, 30 s), and extension (72 C, 30 s). The amount of DNA or number of hUCBMSCs, was calculated by fitting the respective threshold cycle (CT)
Please cite this article in press as: H.S. Kim, et al., Lowering the concentration affects the migration and viability of intracerebroventriculardelivered human mesenchymal stem cells, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.115
Fig. 1. MR images of ICV-injected ferumoxytol-labeled hUCB-MSCs with varying concentrations. T2 weighted and T2* MR images reveal the presence of hypo-intense signals (indicated by red arrows) in the lateral ventricles for all three concentration groups (A) high, (B) middle, and (C) low. The higher the concentration, the MR signal seemed more pronounced. Signals were also detected in the contralateral side for the low concentration group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Histological confirmation of MR images. Prussian blue and immunohistochemical (IHC) stains of sections corresponding to the MR images for the (A) high, (B) middle, and (C) low concentration groups. The blue (prussian blue) and human mitochondria (IHC) stains confirmed the presence of ferumoxytol-labeled hUCB-MSCs at the site of hypointensity. Cellular distribution is greater with decrease in cell concentration. Prussian blue: scale bar ¼ 1 mm (left), 100 mm (right), IHC: scale bar ¼ 100 mm.
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known to have an important role in migration [17], total RNA was isolated from the samples using TRIzol (Invitrogen, MA, USA). cDNA was synthesized from 1000 ng of RNA from each of the samples by following the manufacturer's instructions (Superscript III Reverse Transcriptase; Invitrogen, USA). PCR cycling conditions (40 cycles) were carried out as follows: initial hold (95 C, 10 min), denaturation (95 C, 15 s), annealing (60 C, 1 min), to amplify the 20 ml reaction mixtures. Analysis of relative gene expression data was carried out by using the comparative CT method [18]. 2.8. CCK8 and LDH assay The in vivo experiment was recapitulated in vitro where the equivalent number and concentration of cells for each group was injected into ultra-low attachment 96 well plate (Costar, NY, USA) wells (in triplicates) containing 100 ml of CSF at the equivalent rate of 0.5 ml per min by using the same stereotactic system used for the animal experiment. After incubation in 37 C, 5% CO2 for a day, cell viability was measured using the cell counting kit-8 (CCK8, Dojindo Laboratories, Japan). Lactate dehydrogenase activity was evaluated based on previously reported procedures [19,20]. The fluorescence of both of the assays was read using a microplate spectrophotometer (Bio-rad, CA, USA). CSF only wells (in triplicates) were prepared for background subtraction for both assays. 2.9. Statistical analysis All values are represented as mean ± standard error of mean (S.E.M). One-way ANOVA (Tukey post-hoc) and paired t tests were used to assess significance and a p-value 0.05 was considered statistically significant (GraphPad Software, CA, USA). 3. Results 3.1. Effects of cell concentration on distribution of ferumoxytollabeled hUCB-MSCs based on MRI
Fig. 3. Quantitative assessment of the anatomical distributions of hUCB-MSCs in the mouse brain. (A) Schematic illustration showing the four different 2 mm coronal brain sections (regions 1, 2, 3, 4) that were acquired and analyzed for real-time PCR. (B) Plotted are the average numbers of MSCs present within each of the respective brain sections for the following groups: control, high, middle, and low. Compared to the high concentration group, a significant difference existed in the average number of engrafted MSCs for the middle and low concentration groups. (C) Total number (average) of residual cells remaining in the brains (sum of the four regions) of mice from each of the groups. Decreasing the concentration resulted in a greater loss of cells. All data are shown as mean ± S.E.M.; *p < 0.05 vs. the high group.
values to a standard curve that was created by varying the number of hUCB-MSCs (102, 103, 104, 105, and 106) mixed with mouse peripheral blood mononuclear cells (PBMCs) to make a total of 1 106 cells. To observe changes in mRNA expression of the C-X-C chemokine receptor type 4 (CXCR4) gene (forward: 50 -GAG TCG ATG CTG ATC CCA AT-30 , reverse: 50 -AAG GCT ATC AGA AGC GCA AG-30 ), a gene
For each of the groups, ferumoxytol-labeled hUCB-MSCs appeared as dark, hypo-intense signals in the T2 weighted and T2* post MR images (Fig. 1). With increase in cell concentration, the signal appeared focal and more prominent at the site of injection. For example, a dark aggregated mass (red arrows), suggesting the transplanted cell aggregate, was observed in the right lateral ventricle of the high concentration group (Fig. 1A). The size of the predicted transplanted aggregate reduced with decrease in cell concentration according to the T2 MR images (Fig. 1B and C). For the low concentration group, hypo-intense signals were not only detected at the site of transplantation, but also at various regions of the brain including the contralateral side, which indicated a possible increase in cellular distribution following reduction in concentration (Fig. 1C). 3.2. Histological assessment of cellular distribution At the sites where hypo-intense signals were visualized, human MSCs were detected by using the human-specific anti-mitochondria antibody (Fig. 2). The histological stains confirmed that the source of the MR signals was from the transplanted ferumoxytollabeled hUCB-MSCs. Different paraffin sections from each group were selected to identify the localization of MSCs (Fig. 2A). Similar to previous reports, the transplanted MSCs were stained as dark brown, small specks that surrounded the nucleus [15,16]. Although faint, iron positive, blue signals were also observed at the site of MSC engraftment through prussian blue staining. According to the histological stains, the size of the transplanted aggregate cell mass
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was larger near the injection site for the high compared to the middle concentration group (Fig. 2B). Cell aggregates were detected at regions far from the injection site for the low concentration group, which provided further evidence that cell distribution increases with decrease in cell concentration (Fig. 2C). 3.3. Quantitative evaluation on the effects of concentration in cell distribution To quantify the effects of concentration in the overall distribution of ferumoxytol-labeled hUCB-MSCs, the amounts of humanspecific DNA sequences in the mouse brains were quantified by real-time PCR. Four different coronal sections were obtained from each mouse according to Fig. 3A. The higher the concentration, the
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number of human cells significantly increased at the site of injection, region 2 (Fig. 3B). The total number of MSCs was 2.2 and 4.9folds greater when comparing high vs. middle and low concentrations, respectively. Although not as striking as the high group, a greatest number of MSCs was also observed in region 2 for the middle group. For the low concentration group, however, the number of human MSCs varied among the coronal brain sections. Interestingly, unlike the high and middle groups, the greatest number of human cells was detected in region 1 for the low group (Fig. 3B). When assessing the total number of cells present for each group, even though a more widespread distribution of the cells was achieved, decreasing the concentration attributed to an overall loss of cell number (Fig. 3C).
Fig. 4. Assessment of the effects of concentration on cell viability and migration. (A) Confocal images (scale bar ¼ 20 mm) and (B) quantitative analysis of TUNEL-positive cells for each group. TUNEL assay was performed using adjacent sections remaining after histological staining. The confocal images represent higher magnifications of the DAB images shown in box insets on the left. The yellow, broken line indicates the location of the MSC aggregate. Results are represented as mean ± S.E.M. of >3 random fields per group; **p < 0.01. In vitro evaluation of cell death carried out by using the (C) CCK8 and (D) LDH assays. Results are represented as mean ± S.E.M. of three independent experiments; *p < 0.05. (D) Relative gene expression levels of CXCR4. Results are represented as mean ± S.E.M. of three independent experiments; ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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3.4. Assessment of differences in cell viability and migration
Author contribution
Since the overall trend in cellular distribution has been confirmed, for the experiments carried out from here on, differences in cell viability and migration were compared between the high and low concentrations. To evaluate the viability of transplanted MSCs in vivo, adjacent sections remaining after immunohistochemical staining were selected to perform the TUNEL assay (Fig. 4A). Overall, the percentage of TUNEL-positive cells was below 20%, but a striking and statistically significant difference was noted between the high and low concentration groups (Fig. 4B). An in vitro study was also additionally performed to recapitulate the in vivo results. MSCs were injected into 96-well plate wells containing CSF to mimic the injection of cells into the lateral ventricle in vivo. Compared to the TUNEL assay, similar trends were observed from the CCK8 (Fig. 4C) and LDH (Fig. 4D) assays. The low concentration group showed the highest viability (1.87-fold increase compared to the high concentration group) and lowest LDH release (1.38-fold increase in LDH release was evident for the high concentration group), respectively. Interestingly, CXCR4 expression was 1.5-fold higher for the low concentration group, thus highlighting enhanced migratory abilities with decrease in cell concentration (Fig. 4E).
HSK and NKL equally performed the overall experiments. HSK, NKL, JWC, and DLN designed and conceived the study. DKY acquired the MR images. JML collected and stored the CSF samples. SJC and WIO received permission to use the hUCB-MSCs. HSK, NKL, JWC, and DLN wrote and revised the manuscript. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment We would like to thank Jong Sin Park for providing help with the graphical illustrations. This study was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C3484), the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C2746), and Basic Research Program through the National Research Foundation of South Korea (NRF) funded by the Ministry of Education (NRF-2014R1A2A1A11050576). Transparency document
4. Discussion Other than widespread distribution, ICV administration has low surgical trauma and MSCs delivered through this route are also capable of activating stem cells residing in the subventricular zone [21e23]. The barrier between the brain parenchyma and ventricle is more permeable than that of the blood brain barrier (BBB), thus increasing the changes of MSC penetration into the brain parenchyma [24]. In one of our recent studies, we have observed the migration of MSCs into the brain parenchyma of a canine model when administered via the ICV route. Based on review of prior studies, varying concentrations such as 2.5 105 cells in 2 ml or 5 104 cells in 1 ml (both are higher than the concentration of the “high” group in this study) were used when performing ICV injections [9e11]. Although the feasibility of the ICV route in delivering various concentrations of cells have been diversely reported, how varying cell concentration affects the overall physiological state of MSCs in terms of migration and viability have not been closely investigated. According to the results of this study, MSCs were observed at the site of injection as well as various regions of the brain for all three concentrations (high, middle, and low). The lower the concentration, a more widespread distribution of the MSCs was noted. Contrastingly, MSCs delivered with a high concentration showed higher localization near the site of injection. The low concentration group also showed the highest viability. Such results are indicative that MSCs injected at low concentrations are more capable of exerting therapeutic action through secretion of paracrine factors in comparison to cells injected at higher concentrations. Paracrine factors secreted by MSCs, however, may encounter dilution effects from the surrounding CSF. Additionally, loss in total number of cells is highest for the low concentration group. Thus, although widespread migration can be achieved, when injected at low concentrations, the overall number of remaining cells after washout will be too low to generate therapeutic benefits. This loss, however, can be compensated by performing multiple injections. For future studies, considering such factors will serve beneficial roles when applied to numerous diseases such as AD where pathology is not localized.
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Please cite this article in press as: H.S. Kim, et al., Lowering the concentration affects the migration and viability of intracerebroventriculardelivered human mesenchymal stem cells, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.08.115