Intravenous administration of iPS‐MSCSPIONs mobilized into CKD parenchyma and effectively preserved residual renal function in CKD rat

Abstract This study traced intravenously administered induced pluripotent stem cell (iPSC)‐derived mesenchymal stem cells (MSC) and assessed the impact of iPSC‐MSC on preserving renal function in SD rat after 5/6 nephrectomy. The results of in vitro study showed that FeraTrack™Direct contrast particles (ie intracellular magnetic labelling) in the iPSC‐MSC (ie iPS‐MSCSPIONs) were clearly identified by Prussian blue stain. Adult‐male SD rats (n = 40) were categorized into group 1 (SC), group 2 [SC + iPS‐MSCSPIONs (1.0 × 106cells)/intravenous administration post‐day‐14 CKD procedure], group 3 (CKD), group 4 [CKD + iPS‐MSCSPIONs (0.5 × 106cells)] and group 5 [CKD + iPS‐MSCSPIONs (1.0 × 106cells)]. By day‐15 after CKD induction, abdominal MRI demonstrated that iPS‐MSCSPIONs were only in the CKD parenchyma of groups 4 and 5. By day 60, the creatinine level/ratio of urine protein to urine creatinine/kidney injury score (by haematoxylin and eosin stain)/fibrotic area (Masson's trichrome stain)/IF microscopic finding of kidney injury molecule‐1 expression was lowest in groups 1 and 2, highest in group 3, and significantly higher in group 4 than in group 5, whereas IF microscopic findings of podocyte components (ZO‐1/synaptopodin) and protein levels of anti‐apoptosis ((Bad/Bcl‐xL/Bcl‐2) exhibited an opposite pattern to creatinine level among the five groups (all P < .0001). The protein expressions of cell‐proliferation signals (PI3K/p‐Akt/m‐TOR, p‐ERK1/2, FOXO1/GSK3β/p90RSK), apoptotic/DNA‐damage (Bax/caspases8‐10/cytosolic‐mitochondria) and inflammatory (TNF‐α/TNFR1/TRAF2/NF‐κB) biomarkers displayed an identical pattern to creatinine level among the five groups (all P < .0001). The iPS‐MSCSPIONs that were identified only in CKD parenchyma effectively protected the kidney against CKD injury.

In the normal physiological state, sufficient tissue stem cells or circulating progenitor cells should be competent to repair or regenerate minor injuries of the renal tubules/kidney parenchyma. [17][18][19] However, in the setting of CKD, renal functional deterioration is faster than the intrinsic repair mechanisms. Accordingly, exogenous help for endogenously tissue regeneration may be a feasible method to rebuild the architectural integrity of kidney.
Interestingly, pre-clinical and clinical studies 18,[20][21][22][23][24] have shown that therapy with mesenchymal stem cells (MSCs)/endothelial progenitor cells (EPCs) for CKD is safe and preserves residual renal function in the setting of CKD. Recently, human induced pluripotent stem cell (iPSC)-derived MSCs have been shown to exhibit multiple paracrine actions for organ repair and regeneration because of the strong capacity of self-renewal and differentiation into most somatic cell lineages. 25,26 Additionally, our previous study 27 also showed iP-SC-derived MSCs therapy effectively protected the rat kidney from acute ischaemia-reperfusion injury. Furthermore, as compared with other MSCs, iPSC-MSCs have great potential for differentiation, proliferation and self-expansion. Moreover, its advantage is that it could always supply adequate number of allogenic MSCs for clinical application as a result of the generation of iPSC-MSC platform has been well created by scientists.
Intriguingly, the fate of intravenous stem cells used to treat the chronic stage of ischaemic-related organ dysfunction, including CKD, has not been elucidated. Magnetic resonance imaging (MRI) offers high-resolution visualization of the fate of cells after transplantation and evaluation of cell-based repair, replacement and therapeutic strategies. Several paramagnetic contrast agents have been successfully used for in vivo cell tracking by MRI. 28,29 Accordingly, the aims of the present study were to assess, using a CKD model and MRI examination, the impact of iPS-MSCs therapy on preserving residual renal function, the signalling pathways and the final destination of iPS-MSCs after intravenous administration.

| Ethics
All animal procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital

| Cell culture for assessing the impact of iPS-MSC treatment on protecting NRK-52E cells against oxidative-stress damage
To elucidate the impact of iPS-MSCs on transferring mitochondria to oxidative-stress damaged normal rat kidney epithelial cells IF microscopic findings of podocyte components (ZO-1/synaptopodin) and protein levels of anti-apoptosis ((Bad/Bcl-xL/Bcl-2) exhibited an opposite pattern to creatinine level among the five groups (all P < .0001). The protein expressions of cell-proliferation signals (PI3K/p-Akt/m-TOR, p-ERK1/2, FOXO1/GSK3β/p90RSK), apoptotic/ DNA-damage (Bax/caspases8-10/cytosolic-mitochondria) and inflammatory (TNF-α/ TNFR1/TRAF2/NF-κB) biomarkers displayed an identical pattern to creatinine level among the five groups (all P < .0001). The iPS-MSC SPIONs that were identified only in CKD parenchyma effectively protected the kidney against CKD injury.

K E Y W O R D S
apoptosis, chronic kidney disease, induced pluripotent stem cells-derived mesenchymal stem cells, inflammation, magnetic characterization of iron oxide, nanoparticles (NRK-52E cells; a rat renal proximal tubular cell line) (Bioresource Collection and Research Center), a transwell assay was utilized for in vitro study. Briefly, NRK-52E cells were pre-treated with H 2 O 2 (200 µmol/L) for 3 hours followed by washing and then were seeded in the bottom of the chamber (Figure 2). The iPS-MSCs suspension was placed in the upper compartment and cocultured with the NRK-52E cells for 6 hours. Following this, the NRK-52E cells were collected to assess for transfer of mitochondrial ability from iPS-MSCs to NRK-52E cells. NRK-52E cells were collected to determine the relative number of mitochondrial DNA in NRK-52E cells as well as the protein levels of inflammatory, apoptotic, oxidative-stress and mitochondrial-damaged biomarkers in these cells.

| Procedure and protocol of cell culture for differentiation of human iPS into MSCs
The procedure and protocol of human iPSC culture for differentiation into MSCs were as per the manufacturer's instructions and based on our previous report ( Figure 1). 27 In detail, by day 1, human iPSCs [mTeSR™1; StemCell, #28315) were first washed by 5 mL PBS, followed by 2 mL Accutase (Gibco, #A1110501; Accutase: PBS = 1:1); the incubator reaction was continuous for 1 minute.
Additionally, 2 mL KO DMEM/F12 (Gibco, #12660012) was added and the cells were collected in 15 mL centrifuge tubes for 5 minutes centrifugation (×200 g). The cells were then cultured in a 10 cm dish for 24 hours in mTeSR™1 culture medium.
By day 2, the cells (mTeSR™1) were collected and washed with 5 mL PBS. STEMdiff TM -ACF Mesenchymal Induction Medium (StemCell, #05241) was added, and the incubator culture was continued for 24 hours. The STEMdiff™-ACF Mesenchymal Induction Medium was exchanged once/day from days 1 to 3. This procedure was repeated on days 3 to 6. On days 7 to 21, the procedure was repeated but the culture medium was refreshed every 3 days.
At 30 minutes prior to transfusion of the iPSCs-MSCs to the animals, the cells were stained by Cell tracker (Molecular probes), that is for tracking iPSCs-MSCs in the renal tissues with a red colour under the immunofluorescent microscopic finding.

| Human iPS-derived MSCs were instructed to differentiate into adipocytes, chondrocytes, and osteoblasts
The procedure and protocol have been described by our previous study ( Figure 1). 27

| Assessment of blood urine nitrogen (BUN) and creatinine levels
To determine whether the animal model of CKD was successfully created, blood samples were serially collected before and after the CKD procedure (ie prior to and at days 14 and 60 before the animals were killed). Serum levels of creatinine and BUN were measured in duplicate using standard laboratory equipment. The mean intra-assay coefficient of variance for BUN and creatinine was less than 4.0%.

| Collection of 24-hr urine for the ratio of urine protein to creatinine at baseline and at days 14 and 60 after CKD Induction
The procedure and protocol have been described in our previous report. 22  water. Urine in 24 hours was collected in all animals prior to and at days 14 and 60 after CKD induction for determining the ratio of urine protein to urine creatinine.

| The iPS-MSCs labelled with iron oxide and Prussian Blue staining
The Iron-labelled iPS-MSCs, at a confluence of 80%, were incu-

| Cell tracing by magnetic resonance imaging (MRI)
The procedure and protocol for renal MRI examination has been reported by our previous study in detail. 22

| Western blot analysis of left kidney specimens
The procedure and protocol have been described in detail in our previous reports. 20 Signals were detected with horseradish peroxidase (HRP)-conjugated goat antimouse, goat anti-rat, or goat anti-rabbit IgG.
Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences), which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP). For oxyblot protein analysis, a standard control was loaded on each gel. F I G U R E 1 Flow cytometric analysis and IF microscopic findings for identification the feature of iPS and iPS-MSC surface markers, and cell-culture results of iPSC-derived MSCs differentiated into adipocytes, chondrocytes and osteocytes. A, Indicated isotype of flow cytometric analysis (ie negative control). B to D, Illustrating the flow cytometric analysis for identifying the expression of induced pluripotent stem cell (iPS) surface makers of SSEA4 + (B), Nanog + (C) and Oct3/4 + (D) cells. E to P, Illustrating the immunofluorescent (IF) microscopic finding (400x) for identify the morphological features of iPS during cell culturing and their distinctive surface markers of positively stained OCT-4, SSEA4, TRA-1-60 and TRA-1-80. M to P, Expressed the merge results of Alexa Fluor488 and Hoechst33342 of these four iPS cells. Q1 to Q5, Illustrating the flow cytometric results of cell culture for identification of percentage of CD90 + , CD44 + , CD105 + , CD73 + and CD271 + , five typical iPS-derived mesenchymal stem cell (MSC) surface markers. Q6, Indicated the negative control of flow cytometric analysis. 1-R1 to R4, Illustrating the microscopic finding of immunofluorescent (IHC) stain for identification of iPSC-derived MSCs differentiated into adipocytes. S1 to S3, Illustrating the microscopic finding of IHC stain for identification of iPSC-derived MSCs differentiated into osteocytes. T1 to T4, Illustrating the microscopic finding of IHC stain for identification of iPSC-derived MSCs differentiated into chondrocytes | 3599 SHEU Et al.

| Immunohistochemical and immunofluorescent studies
The procedures and protocols for immunofluorescent (IF) examinations were based on our previous reports. 20

| Statistical analysis
Quantitative data are expressed as means ± SD. Statistical analyses were performed using SAS statistical software for Windows version 8.2 (SAS Institute). ANOVA was conducted followed by Bonferroni multiple comparison post hoc test for comparing variables among groups.
A probability value < .05 was considered statistically significant.

| Flow cytometric analysis and IF microscopic findings for identification typical feature of iPS and iPS-MSC surface markers, and cell-culture results of iPSC-derived MSCs differentiated into adipocytes, chondrocytes and osteocytes
The cell surface makers of SSEA4 + , Nanog + and Oct3/4 + , three typical iPS surface markers, were clearly identified by flow cytometric analysis with an estimated expression of more than 95% ( Figure 1B-D).

| Impact of iPS-MSC treatment on protecting the NRK-52E cells against oxidative-stress damage
Relative mitochondrial DNA copy number was significantly lower in NRK-52E + H 2 O 2 (ie group B) than in the control group (ie group A), than were reversed in NRK-52E + H 2 O 2 + iPS-MSCs (ie group C), suggesting that the iPS-MSCs were able to transfer mitochondria into oxidative-stress injured NRK-52E cells (Figure 2A).
The protein expression of inflammatory (TNF-α, MMP-9), apoptotic (cleaved caspase 3, cleaved PARP, oxidative-stress (NOX-1, NOX-2) and mitochondrial-damaged (cytosolic cytochrome C) biomarkers was significantly increased in group B than in groups A and C and significantly increased in group C than in group A ( Figure 2B-G).

| In vitro and in vivo studies to identify paramagnetic contrast agents labelling the iPS-MSCs and in vivo cell tracing by MRI in living animals
In the in vitro study, the FeraTrack™Direct Kit (ie containing contrast particles) was utilized to test whether the paramagnetic contrast agents Interestingly, among these three groups, the signalling of iPS-MSC S-PIONs was detected by MRI examination (ie in living animals) and HE, stain (ie in kidney specimen by day 60 after CKD induction) only in groups 4 and 5 animals but not in group 2 animals ( Figure 3E-I), highlighting that the iPS-MSCs preferentially mobilized from circulation to damaged kidney (ie CKD) than to healthy kidney (ie SC) for tissue regeneration and organ repair.

| The time courses of circulating levels of BUN and creatinine and ratio of urine creatinine to urine protein (R uP/uCr )
By day 0 (ie at baseline), the BUN and creatinine level and the R uP/uCr did not differ among the five groups ( Figure 4A-C). However, by day 20 after CKD induction these three parameters were significantly increased in group 5 and further significantly increased in groups 3 (ie CKD only) and 4 and 5 when compared to groups 1 (ie SC) and 2, but they showed no difference between groups 1 and 2 or between groups 3 and 4 ( Figure 4D-F). Furthermore, by days 40 and 60 after CKD induction (ie end of study period), these three parameters were highest in group 3, lowest in groups 1 and 3, and significantly higher in group 4 than in that of group 5 but they showed no difference between groups 1 and 2 ( Figure 4G-L).
Regarding pathological findings ( Figure 4M-Q), the kidney injury score was highest in group 3, lowest in groups 1 and 2 and significantly higher in group 4 than in group 5, but it displayed no difference between groups 1 and 2 ( Figure 4R).

| Immunofluorescent microscopic findings of kidney parenchyma by day 60 after CKD induction
The cellular expressions of ZO-1 and synaptopodin, two components of podocytes, were highest in groups 1 and 2, lowest in group 3 and significantly lower in group 4 than in group 5, but these two parameters did not differ between groups 1 and 2 ( Figure 5).
Additionally, the cellular expression of CD31, an endothelial cell (EC) surface maker indicating the integrity of EC, exhibited an identical pattern of ZO-1 among the five groups ( Figure 6). Conversely, the cellular expression of KIM-1, a typical biomarker of renal tubular injury, showed an opposite pattern to ZO-1 among the five groups ( Figure 6).

| Protein expressions of inflammatory cell apoptotic, cell death/proliferation signalling pathways in kidney parenchyma by day 60 after CKD induction
The MAPK-activated protein kinase-1 (p90RSK), which regulate CREB, 10 cell proliferation/survival signalling (Figure 9), displayed an identical pattern to apoptosis among the five groups.

| Kidney injury score and fibrotic area in kidney parenchyma by day 60 after CKD induction
To elucidate the degrees of kidney injury and fibrosis in kidney parenchyma, the HE, stain and Masson's trichrome stain were utilized in the present study, respectively. As expected, these two parameters were highest in group 3, lowest in groups 1 and 2 and significantly higher in group 4 than in group 5, but these biomarkers did not differ between groups 1 and 2 ( Figure 10). been fully investigated. This raises questions why some investigators in clinical trials tend towards intra-arterial administration of stem cells into ischaemic organs for improving ischaemia-related organ dysfunction, even though intra-arterial administration of stem cells is more invasive than intravenous administration. 23,32,33 The novel finding in the present study was that, using MRI examination, we precisely identified that intravenously administered iPS-MSC SPIONs (ie iron-labelled iPS-MSCs) settled in kidney parenchyma of CKD, but not in healthy, kidney, highlighting that a chronically dysfunctional organ can capture stem cells for tissue/organ repair and regeneration. Accordingly, our data provide important and clinically supportive information for intravenous administration (ie an alternative route) of stem cell therapy.

| D ISCUSS I ON
Numerous data have emphasized the therapeutic impact of stem cells on improving ischaemic-related organ dysfunction. 18,[20][21][22][23][24] The most important finding in the present study was that, as compared with CKD only, intravenous administration of iPS-MSCs significantly preserved residual renal function (ie reduced the levels of creatinine/ BUN and the ratio of urine creatinine to urine protein) and reduced the kidney injury score (ie HE, stain pathological findings). Our findings corroborate those of previous studies. 18,[20][21][22][23][24] Additionally, the cellular expressions of podocyte components (ie ZO-1, synaptopodin) and endothelial cell surface marker (ie CD31 + ) were preserved, whereas renal tubular injury biomarker (ie KIM-1) was substantially reduced in CKD animals treated by iPS-MSCs than in CKD only animals. This could, at least in part, explain why proteinuria is much higher in CKD animals without, than in with, iPS-MSCs therapy.
An association among acute inflammation, apoptosis/cell death and organ damage have been extensively investigated. 20,22,27,34 An essential finding in the present study was that the acute inflammatory and mitochondrial-damaged biomarkers were markedly increased in CKD animals compared to SC animals. Another essential finding in the current study was that protein levels of apoptosis in kidney specimens were significantly increased in CKD animals compared with SC animals. However, these molecular perturbations were substantially reduced by low-dose, and further substantially by high-dose, iPS-MSCs treatment. In this way, our findings strengthen those from previous studies 20,22,27,34 and once again confirmed that the intravenous route adequately supplied iPS-MSCs for improving the outcome of rat CKD.
Numerous cell-stress signalling pathways are frequently initiated by acute cell/organ damage with the aim of cell proliferation/ cell survival and death. 35 In the present study, we found that several cell-stress signalling pathways, including PI3K/Akt/m-TORAkt substrates (FoxO1/GSK3β), p90RSK (ie MAPK-activated protein kinase-1 for regulating CREB), p-ERK1/2 and p-PKC, were activated in CKD animals, suggesting intrinsic responses for ischaemia/stress stimulation. However, these signalling pathways were down-regulated by iPS-MSCs therapy, implicating that the cellular intrinsic responses to stress/ischaemic stimulation were relieved by cell therapy.

| Study limitation
This study has limitations. First, the MRI examination occurred the day after iron-labelled iPS-MSCs were intravenously administered; therefore, the long-term fate of these cells is currently unclear. Second, only qualitative rather than quantitative measurement was provided by MRI examination. We therefore do not know how many cells entered the kidney and how many were trapped in other organs, notably the lung.

| CON CLUS ION
The results of the present study demonstrated that iron-labelled iPS-MSCs were identified in the parenchyma of CKD animals and effectively protected the kidney architecture and residual renal function against CKD deterioration.

ACK N OWLED G EM ENTS
This study was supported by a programme grant from Chang Gung Memorial Hospital, Chang Gung University (Grant number: CMRPG8G1291).

CO N FLI C T O F I NTE R E S T
All authors have read the journal's policy on disclosure of potential conflicts of interest and the journal's authorship agreement. The authors declare that they have no conflicts of interest. The article has been reviewed by and approved by all named authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding authors upon reasonable request.