Iron Sucrose-Labeled Human Mesenchymal Stem Cells: In Vitro Multilineage Capability and In Vivo Traceability in a Lapine Xenotransplantation Model

Abstract

For evaluation of cell therapy applications, it is of interest to be able to trace and observe cellular distribution of the transplanted cells. The aim with the study was to examine viability, traceability, and multilineage capability of iron sucrose-labeled mesenchymal stem cells (MSCs) after transplantation into lapine intervertebral discs (IVDs). MSCs were collected from three human donors, age 31–50 years, and IVDs from 12 rabbits, age 3 months. MSCs were isolated from the bone marrow and cultured using standard protocols. Iron sucrose labeling of MSCs was performed in Dulbecco's Modified Eagle's Medium-low glucose with Venofer^®. The iron sucrose-labeled MSCs were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. Results were evaluated using Oil red, von Kossa, Alcian blue, and collagen II (immunohistochemistry). For the animal experiments, iron sucrose-labeled MSCs and nonlabeled MSCs were injected into lapine IVDs (LI-LIV level). After transplantation, at the time points of 1 and 3 months, IVDs were collected and cells were analyzed for cell viability (fluorescence-activated cell sorting). The lapine IVDs were collected and examined for presence of cells positive for iron deposits using Berliner blue staining. Differentiation of the iron sucrose-labeled MSCs into adipogenic (lipid droplets), osteogenic (calcium deposits), and chondrogenic lineage (proteoglycan/collagen II accumulation) (3/3 donors) was observed in vitro. After transplantation, the mean cell viability for iron-labeled MSCs/IVD cells was 99%, for nonlabeled MSCs/IVD cells was 95%, and for control IVD cells was 99% at a time point of 1 month. At a time point of 3 months, mean cell viability was 73% for iron sucrose-labeled MSCs/IVD cells, for nonlabeled MSCs/IVD cells was 77%, and for control IVD cells was 98%. At the time point of 1 month, cells positive for iron deposits were detected sparsely distributed in IVDs (tissue sections) in 4/4 animals and at the time point of 3 months in 4/4 animals. The results indicate that iron sucrose can be used as a cell tracer with a stable detection potential in tissues (histologies). This may be an important evaluation tool for understanding stem cell distribution/function after transplantation into degenerated cartilaginous tissues.

Introduction

At present, regenerative medicine is a research field worldwide that escalates in an increasing pace. Different types of biological applications involving cell therapy using mesenchymal stem cells (MSCs) are under examination and certain applications are already in clinical use [1,2]. At the present time, both local and systemic administration applications are used to transplant MSCs into different organs [1,3 –5]. For evaluation of novel cell therapy applications, for example, transplantation of MSCs into degenerated intervertebral discs (IVDs) would be of benefit to be able to trace the transplanted cells. Iron sucrose has been proposed as a possible (intra) cellular compound for cell tracer purpose [6].

Furthermore, it would be optimal to be able to observe localization, migration, and function of transplanted cells. Recently, superparamagnetic iron nanoparticles (SPIOs, Endorem^®), which are nontoxic, have been used clinically for the detection of tumors and metastases in, for example, the lymphatic system using magnetic resonance imaging (MRI) [7]. Furthermore, SPIOs have been used as cell tracers in in vitro studies [8 –11] and in vivo studies (animal models and human) [7,8,12 –14]. Unfortunately, SPIOs that are clinically approved are presently not available in the market. However, other iron compounds could be thought to be a feasible option to function as potential cell tracers. In a recent study, the effect on traceability, viability, and chondrogenic differentiation of human MSCs, which were labeled with iron sucrose, was investigated in vitro. In that study, it was observed that using iron sucrose as a cell tracer only to a minor extent affected the stemness of MSCs and chondrogenic differentiation [6]. In general, one capacity that is critical to the identity of stem cells is to have a correct function to proliferate in a manner that maintains stemness as well as to generate daughter cells that are capable of multilineage differentiation [15,16]. However, there is limited knowledge regarding the effects of iron labeling of, for example, human MSCs on stemness, for example, multilineage capability, as well as on cell viability and traceability in vivo after a stem cell transplantation.

In the present study, the iron sucrose compound, Venofer^®, a pharmaceutical clinically approved drug, commonly used for patients with, for example, iron deficiency [17,18] was examined and the potential capability of this iron compound to be used as a cell tracer in vivo was investigated. The compound Venofer consists of cores of iron (III)-hydroxide, which are superficially surrounded by many noncovalently bound sucrose molecules, resulting in a complex with the molecular mass of ∼34–60 kDa [19]. The iron in this molecular complex is bound in a similar way as the natural physiologically occurring ferritin (intracellular form of iron in humans) [20,21]. The advantage to use iron sucrose as a cell tracer is the nontoxic features of this compound and that it maintains a detectable strong signal over time in, for example, histology samples.

Commonly, a set of accepted stem cell markers is used for characterization and control of the purity of human MSC populations. In this study, the accepted cell surface protein markers CD105, CD166, CD34, and CD45 for profiling of human MSCs were used [22,23]. Furthermore, the human MSCs should be able to differentiate into osteoblasts, chondrocytes, and adipocytes under certain standard differentiation systems in vitro [22 –25] and/or in in vivo models, for example, when injected into immune-deficient mice [26].

The aim of the study was to determine multilineage capability of iron sucrose-labeled human MSCs in vitro and to examine the cell viability and traceability of iron sucrose-labeled human MSCs after xenotransplantation into lapine IVDs.

Materials and Methods

Cells and tissues

Human MSCs

Human MSCs were collected from the bone marrow (iliac crest) of three donors, female, age 31 (donor 1), male, age 44 (donor 2), and male, age 50 (donor 3) years, undergoing spinal surgery (see further below under the section Isolation of human MSCs and monolayer cultures). All experiments were approved by the regional human ethics committee of Sweden (ethical permission no: 532-04) and the samples were collected with informed consent from all the donors.

Animals

In total, 12 female New Zealand white rabbits (Linköpings rabbit farm), age 3 months, were used in the present study. Six animals per time point were sacrificed at the time points of 1 and 3 months after transplantation, and the spines (lumbar level LI–LV) were harvested en bloc. Eight animals were used for immunohistochemistry (IHC) analyses and four animals for the fluorescence-activated cell sorting (FACS) analyses. See further below under the section Cell viability of cells (human and lapine) isolated from lapine IVDs after transplantation.

The animals demonstrated a good health status and gained weight during the full experimental time period. All animal experiments were approved by the Regional Animal Ethics Committee, Vastra Gotaland region, Sweden (ethical permission no: 4-2014).

Isolation of human MSCs and monolayer cultures

The MSCs from three donors were isolated from the bone marrow (iliac crest) in cell preparation tubes (Ficoll) using standard protocols. The MSCs were seeded in cell culture flasks (NUNC, Penfield) in a cell density of 200×10³/cm² and expanded in Dulbecco's Modified Eagle's Medium-low glucose (DMEM-LG) medium (Invitrogen). The cell culture medium was supplemented with 2 mM L-glutamine (Gibco), 0.1 mg/mL penicillin/streptomycin (PAA laboratories), 10 ng/mL β-FGF (Invitrogen), and 10% human serum. The cells were thereafter cultured in an incubator (Heraeus BBD 6220; Thermo Fisher Scientific) (37°C, 5% CO₂, 95% air). At 90% confluence, the MSCs were detached using Trypsin/EDTA solution (Invitrogen), reseeded at the concentration of 15×10³/cm², and thereafter expanded to passage 4–5.

Cell surface marker profile of human MSCs

FACS of the human MSCs (n=3) (passage 3–4) was performed to check the purity of the MSC populations by analysis for a set of accepted human MSC surface markers: CD105, CD166, CD34, and CD45 [23]. The antibodies that were used in the analysis were as follows: CD166-PE (Becton Dickson), CD105-FITC (AbD Serotec), CD45-PE (Becton Dickson), and CD34-FITC (BD Biosciences). Six thousand cells per sample were analyzed using a FACSAria flow cytometer (Becton Dickson). Negative controls were FITC- and PE-conjugated IgG isotype controls (Becton Dickson).

Test of cell viability of human MSCs before transplantation

Human MSCs (n=3) were tested for cell viability before transplantation. The samples were washed with a phosphate saline buffer, incubated for 10 min with 7-aminoactinomycin (7-AAD) solution (BD Biosciences) according to the manufacturer's protocols, and analyzed by flow cytometry using a FACSAria instrument (BD Biosciences). MSCs from donor 1 were used for transplantation into the lapine IVDs. Five thousand cells per sample were analyzed.

Iron labeling of human MSCs

The human MSCs were labeled as in a previously described method [6] with Venofer (Luitpold pharmaceuticals), a polynuclear iron (III)-hydroxide in sucrose (iron sucrose) ([Na₂Fe₅O₈(OH)·3(H₂O)]n·m(C₁₂H₂₂O₁₁)) containing 20 mg elemental iron per milliliter (aqueous solution of brown color). The cell media were removed from the cell cultures, the cells were subsequently washed with DMEM- LG (no serum added), and thereafter serum-free DMEM-LG containing Venofer diluted to 1 mg/mL (1 mg elemental iron/ml) was added to the human MSC cultures. The human MSC cultures were then incubated for 16 h (in 37°C, 5% CO₂, 95% air) before the xenotransplantation (see further below under the section In vivo experiments' xenotransplantation of human MSCs). The dose of 1 mg/mL Venofer used in the iron labeling of the MSCs was based on previous studies for iron labeling of cells in cell cultures [6,9]. For simplicity, the iron sucrose labeling will hereafter be referred to in the following text as iron labeling.

Test of multilineage differentiation capability of iron-labeled MSCs

Differentiation into the chondrogenic lineage

After expansion of the human MSCs in monolayer culture, iron-labeled MSCs from the donors (n=3) (passage 4) were cultured in the pellet mass culture system [27]. For the pellet cultures, 200,000 cells were pipetted into polypropylene conical tubes containing 0.5 mL of a defined chondrogenic medium: Dulbecco's Modified Eagle's Medium-high glucose (DMEM-HG; PAA Laboratories) supplemented with 5.0 μg/mL linoleic acid (Sigma-Aldrich), insulin, transferrin, and selenium (ITS-G; Life Technologies), 1.0 mg/mL human serum albumin (Equitech-Bio), 10 ng/mL TGF-(1 (R&D Systems), 10⁻⁷ M dexamethasone (Sigma Aldrich), 14 μg/mL ascorbic acid (Sigma Aldrich), and 1% penicillin–streptomycin solution (PAA Laboratories). Thereafter, the cells were centrifuged at 500 g for 5 min and cultured in an incubator (37°C, 5% CO₂, 95% air). The medium was changed twice a week. Duplicate pellet cultures were made and the pellets were harvested after 7 and 28 days. Controls were simultaneously cultured pellets with noniron-labeled MSCs from the same donors. The harvested cell pellets were fixated with 4% formaldehyde (Histolab products AB) and embedded in paraffin, and sections of 5 μm thickness were prepared. The sections were stained for expression of glycosaminoglycans and collagen IIA1 and examined using a microscopy NIKONEqlipse600 (NIKON).

Duplicate cell cultures were performed. See further below under the sections Histology: iron staining of cells and tissues; Histology: glycosaminoglycans; and Immunohistochemistry.

Differentiation into the adipogenic lineage

For differentiation into the adipogenic lineage of iron-labeled MSCs, cell cultures from the donors (n=3) were allowed to grow to 90% confluence in the MSC medium (see above under the section Isolation of human MSCs and monolayer cultures) and thereafter were trypsinized and reseeded in 24-well plates at a concentration of 10,000 cells/cm². The cells were cultured in triplicate cultures and incubated in an incubator (37°C, 7% CO₂, 93% air). For differentiation of the MSCs into adipocytes, a STEMPRO^® Adipogenesis Differentiation Kit (Life Technologies) was used supplemented with penicillin and streptomycin (PAA laboratories). The cell cultures were harvested at day 8, and the samples were fixated with 4% formaldehyde (Histolab). Sections were thereafter stained with oil red staining (standard protocols) [28] for the detection of lipid-containing vesicles (red color) in adipocytes by light microscopy.

Differentiation into the osteogenic lineage

For differentiation into the osteogenic lineage of iron-labeled MSCs, isolated cells from the donors (n=3) were cultured in DMEM/LG supplemented with 1% mM ascorbic acid (Sigma-Aldrich), 1% 10⁻⁵ M dexamethasone (Sigma-Aldrich), and 10% human serum.

At day 10, 1% 0.2 M β-glycerophosphate (Sigma-Aldrich) was added to the medium for differentiation into osteocytes. The cell cultures were harvested at day 20, and samples were fixated with 4% formaldehyde (Histolab). Sections were stained with von Kossa staining (standard protocols) [29] and examined for the presence of calcium deposits (brown/black color) reflecting the presence of osteocytes, using light microscopy (NIKON). Triplicate cell cultures were performed.

In vivo experiments' xenotransplantation of human MSCs

The animals were anesthetized before surgery with intramuscular injections of fentanyl/fluanisone (HypnormVR; Janssen) (0.7 mg/kg body weight) and an intraperitoneal injection of diazepam (StesolidVR; Actavis AB), (1.5 mg/kg body weight). Two hundred fifty thousand human MSCs (10 μL cell suspension) (donor 1) labeled with iron sucrose compound (Venofer) or nonlabeled human MSCs (same donor) were injected into the center part of the lapine IVDs (with the help of an image intensifier) using a 22-gauge needle (Fig. 1).

FIG. 1.

X-ray images of a lapine spine showing an injection of human MSCs into an IVD taken with an image intensifier at the time point of the xenotransplantation. A 22-gauge needle size was used for the injections of the human MSCs. IVDs, intervertebral discs; MSCs, mesenchymal stem cells.

One IVD per animal was injected with iron-labeled human MSCs and one IVD per animal was injected with noniron-labeled human MSCs, negative controls were naive undisturbed IVDs. IVDs transplanted with noniron-labeled human MSCs and/or naive undisturbed IVDs served as negative controls. The animals were sacrificed at the time points of 1 and 3 months after transplantation with intraperitoneal administration of an overdose of methylphenobarbital (APL). The spines (lumbar level LI–LV) and surrounding tissues were harvested en bloc.

Preparation of cells and tissues for histology

The harvested lumbar spine tissues were immersed in 4% formaldehyde (Histolab), embedded in paraffin, decalcified by using a 12.5% EDTA solution, and sections (thickness 5–7 μm) were prepared. Paraffin sections were deparaffinized with xylene 2×10 min and rehydrated in 99%, 95%, and 70% ethanol for 5 min in each solution before analysis.

Histology: iron staining of cells and tissues

The cell pellet sections (5 μm thickness) were stained for iron deposits present using the Prussian blue reaction (Berliner blue method), which was performed according to standard protocols [30,31].

The IVD tissues were consecutively coronally sectioned (sections of 5 μm thickness), serially numbered, and every twentieth section was stained for iron deposits using the same standard protocols as above. A minimum of two observations in each animal of iron deposit-positive cells in the transplanted IVDs were considered as a positive result.

The cell pellets and IVD tissues sections were examined by light microscopy (NIKON) for iron deposits (blue color). Negative controls were noniron-labeled cell pellets and naive IVDs (undisturbed), which were analyzed according to the same protocol.

Histology: glycosaminoglycans

For examination of ECM accumulation (glycosaminoglycans), the IVD tissues/ cell sections were stained with the Alcian blue staining method [32,33]. The sections were examined using a light microscope (NIKON).

Immunohistochemistry

Deparaffinization was performed of cell pellet sections as described above under the section Preparation of cells and tissues for histology.

For the detection of collagen IIA1 accumulation in pellets, IHC analyses were performed on sections from the iron-labeled and the noniron-labeled cell pellets. Samples were digested with hyaluronidase from bovine testes, 8000 units/mL (Sigma Aldrich) in phosphate-buffered saline (PBS), and pH 7.4 for 1 h at 37°C. Thereafter, the sections were blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich). The goat anti-collagen IIA1 primary antibody (sc-7763, Santa Cruz, Santa Cruz, USA) diluted 1:100 in PBS containing 3% BSA (Sigma-Aldrich) was applied on the samples, which thereafter were incubated overnight in 4°C. The primary antibodies were visualized using an anti-goat secondary antibody, horseradish peroxidase conjugated (sc-3851, Santa Cruz, Biotechnology Inc., Dallas, TX), diluted 1:200, and the sections were incubated for 2 h in room temperature. An enhancement step was performed using the TSA- direct Cy-3 kit system (Perkin Elmer). Negative controls were isotype controls and/or sections incubated with the primary antibody omitted.

For detection of the transplanted human MSCs in the lapine IVD tissue sections, the sections were blocked with the following: 100 mM TRIS buffer, 0.9% NaCl, pH 7.5 (TBS) containing 0.25% Triton-X100 (Sigma-Aldrich), and 3% BSA for 30 min. Samples were then incubated with the primary antibody, mouse anti-human nuclei (mab1281; Millipore Billerica), dilution 1:50 dissolved in TBS, during 24 h in 4°C. A secondary antibody, 2 mg/mL, goat anti-mouse Alexa fluor 546, (Invitrogen) was added in dilution 1:250, and the sections were incubated in 37°C for 3 h. All the samples were mounted with Prolong Gold antifade media (Invitrogen) containing the nuclear staining compound 4,6 diamino-2-phenylindole (DAPI). The samples were thereafter examined using a fluorescence microscope (NIKON) with NIS-elements software (NIKON).

Cell viability of cells (human and lapine) isolated from lapine IVDs after transplantation

Lumbar IVDs from the animals (n=4), transplanted with iron-labeled MSCs, noniron-labeled MSCs, and naive IVDs from two animals per time point, were collected and thereafter the IVD [both annulus fibrosus (AF) and nucleus pulposus (NP)] tissues were manually cut to small pieces with a scalpel. The IVD tissue pieces were thereafter treated with 0.8 mg/mL collagenase type II (Worthington Biochemicals) diluted in Dulbecco's Modified Eagle's Medium-high glucose:nutrient mixture F-12 (DMEM/F12) cell medium (Invitrogen) and incubated for 24 h in an incubator (37°C, 5% CO₂, 95% air). The primary isolated cells were analyzed by 7AAD staining and flow cytometry as described above under the section Test of cell viability of human MSCs before transplantation. Twenty thousand cells per sample were analyzed.

Results

Cell surface marker profile and cell viability of human MSCs

After expansion in cell cultures of human MSCs to passage 3, the cell surface marker profile MSCs were as follows: donor 1; 98% CD105⁺/CD166⁺, <5% CD34⁺/CD45⁺, donor 2; 97% CD105⁺/CD166⁺, <5% CD34⁺/CD45⁺, and for donor 3; 98% CD105⁺/CD166⁺, <5% CD34⁺/CD45⁺ cells. The cell viability of MSCs was for donor 1: 99%, for donor 2: 98%, and for donor 3: 88% before transplantation (Fig. 2).

FIG. 2.

The plots are displaying the results of the fluorescence-activated cell sorting analysis of the human MSCs from donor 1, female, age 31 years: (A) forward scatter plot, (B) plot of CD105⁺ and CD166⁺ cells, (C) plot of CD34⁻ and CD45⁻ cells, (D) negative isotype control, and (E) table of the cell surface marker profile of the human MSCs and the cell viability of all the donors (n=3).

Results of multilineage capability of human MSCs in vitro

The MSCs isolated from the human donors (n=3) displayed multilineage capability in vitro when driven to differentiate into the chondrogenic, adipogenic, and osteogenic lineages.

Differentiation of MSCs into the chondrogenic lineage

The iron-labeled and the noniron-labeled control pellets formed various morphological shapes, rounded or nonrounded in the pellet mass cell cultures. Iron compound deposits were clearly visible as blue granulae in the cells, and the iron deposit-positive cells were relatively evenly distributed in the cell pellets at day 7 and at day 28 (n=3). Noniron-labeled control cell pellets showed no staining (Fig. 3).

FIG. 3.

Images of iron-labeled cell pellets (blue color) from one of the donors, male, age 44 years (donor 2) at (A) day 7, (B) day 28, (C) noniron-labeled control cell pellet, day 7, and (D) noniron-labeled control cell pellet, day 28. Staining: Berliner blue.

In the iron-labeled cell pellets, a weak positive staining for glycosaminoglycan accumulation was detected at day 7 (2/3 donors) and at day 28 (3/3 donors). In the noniron-labeled control pellets, a higher positive staining for glycosaminoglycan accumulation was detected at day 7 (3/3 donors), peaking at day 28 (3/3 donors) (Fig. 4A–D).

FIG. 4.

Chondrogenesis: Glycosaminoglycan accumulation (blue color, white arrowheads) displaying chondrogenic differentiation of MSCs (donor 2, male, age 44 years) in (A, C) iron-labeled cell pellets at day 7 and day 28 and (B, D) noniron-labeled control cell pellets at day 7 and day 28. Staining: Alcian blue. Images of immunohistochemistry results of collagen IIA1 (yellow color, white arrowheads) (marker for early formed ECM in chondrogenesis) stainings in human cell pellets (donor 3, male, age 50 years) in (E, G) iron-labeled cell pellets at day 7 and day 28 and (F, H) noniron-labeled control cell pellets at day 7 and day 28. Nuclei stained with DAPI (blue color). Adipogenesis: images of adipocytes containing vesicles with lipid droplets (red, black arrowheads) detected at day 8 in MSC (donor 1, female age 31 years) cultures driven into the adipogenic lineage: (I) iron-labeled MSCs/cells and (J) noniron-labeled control MSCs/cells. Staining: oil red. Osteogenesis: images of MSC (donor 3, male age 50 years) cultures with calcium deposits (brown/black color, white arrowheads) detected at day 20, reflecting osteogenic differentiation of MSCs: (K) iron-labeled MSCs/cells and (L) noniron-labeled control MSCs/cells. Staining: von Kossa. DAPI, 4,6 diamino-2-phenylindole.

Expression of collagen IIA1 was detected in the iron-labeled pellets at day 7 (3/3 donors) and peaking at day 28 (3/3 donors) in comparable levels to the noniron-labeled control pellets. Negative controls showed no staining (Fig. 4E–H).

Differentiation of MSCs into the adipogenic lineage

In MSC cultures (n=3), adipocytes containing vesicles with lipid droplets were detected at day 8 in similar levels as in the noniron-labeled control cell cultures (Fig. 4I, J).

Differentiation of MSCs into the osteogenic lineage

Calcium deposits reflecting osteogenic differentiation of human MSCs were detected in cell cultures (n=3) at day 20, in comparable levels in the iron and noniron-labeled cell cultures (Fig. 4K, L).

Presence of transplanted human cells in lapine IVDs

The transplanted human iron-labeled MSCs and the noniron-labeled MSCs were detected in the lapine IVDs (ex vivo) in seven of eight animals by IHC stainings as solitary cells and/or in cell foci formations. Naive control IVDs showed no staining (Fig. 5).

FIG. 5.

Immunohistochemistry results of anti-human nuclei stainings. Images of lapine IVDs displaying (A) human iron-labeled MSCs detected 1 month after transplantation, (B) human iron-labeled MSCs detected 3 months after transplantation, (E) human noniron-labeled MSCs detected 1 month after transplantaion, (F) human noniron-labeled MSCs detected 3 months after transplantation, (I, J) images of lapine naive control IVDs (untreated) after 1 and 3 months. Histology images of lapine IVDs transplanted with (C) iron-labeled human MSCs at the time point of 1 month after transplantation, (D) iron-labeled human MSCs at the time point of 3 months after transplantation, (G) noniron-labeled human MSCs in lapine IVDs 1 month after xenotransplantation, (H) noniron-labeled human MSCs in lapine IVDs 3 months after transplantation, (K) naive untreated lapine control IVD at a time point of 1 month, and (L) naive untreated lapine control IVD at a time point of 3 months. Iron particles are visible as small brown dots in the cytosol of the iron-labeled MSCs (C, D). Images were taken in the center of the lapine IVD. Enlarged images in upper right corners. (Arrowheads indicate human cells positive for anti human nuclei staining).

Areas that stained positive for glycosaminoglycans were observed at all time points in close proximity to the transplanted iron-labeled and the noniron-labeled human MSCs in the lapine IVDs in eight of eight animals. In the iron-labeled MSCs, the iron particles were visible as small brown dots. No major differences were observed in ECM accumulation in the IVDs transplanted with iron-labeled or noniron-labeled samples compared to the naive IVDs (Fig. 5).

Traceability of transplanted human iron-labeled MSCs in lapine IVD

Cells positive for iron deposits with a migratory phenotype, elongated and with cellular protrusions, were observed in lapine AF and NP regions as solitary cells and/or in a small cluster (two to five cells per cluster). At the time point of 1 month after transplantation, cells positive for iron deposits were detected sparsely distributed in four of four animals in the AF and the NP. At the time point of 3 months after transplantation, cells positive for iron deposits were detected sparsely distributed in the AF in four of four animals and in the NP in two of four animals. In the control IVD sections from naive as well as in sections of IVDs injected with noniron-labeled MSCs, no cells positive for iron deposits were detected (Fig. 6).

FIG. 6.

Images of human MSCs positive for iron deposits in lapine IVDs 1 month after xenotransplantation (A) in the annulus fibrosus (AF), (B) in the nucleus pulposus (NP), (C) in the AF, and 3 months after xenotransplantation (D) in the NP, (E) in the AF, and (F) in the AF. Enlarged images in upper right corners. (Arrowheads indicate human cells positive for iron deposits). Images of native controls (negative controls) (G) in the AF and (H) in the NP. Staining: Berliner blue.

Cell viability of human cells and lapine IVD cells after xenotransplantation

Lapine IVDs (n=4) transplanted with human MSCs and IVDs (n=4) transplanted with noniron-labeled MSCs were collected from the animals at the time points of 1 and 3 months after transplantation; the cells were isolated and analyzed for cell viability (primary isolated cells). At the time point of 1 month after transplantation, the mean viability of all cells isolated from the lapine IVDs transplanted with human iron-labeled MSCs was 99%, in noniron-labeled MSC transplanted IVDs 95% and in naive lapine IVDs 99%, when analyzed by flow cytometry. Three months after transplantation, the mean viability of cells isolated from the lapine IVDs transplanted with iron-labeled MSCs was 73%, in noniron-labeled MSCs transplanted with lapine IVD 77%, and in naive IVD 98% (Fig. 7).

FIG. 7.

The bar graph displays the mean±2 SEM cell viability of cells isolated from lapine IVDs transplanted with human iron-labeled MSCs (light gray colored bars), noniron-labeled MSCs (dark gray colored bars), and naive lapine IVDs (black colored bars) at the time points of 1 month and 3 months after xenotransplantation.

Discussion

In this study, the results demonstrate that iron-labeled human MSCs were able to differentiate into the chondrogenic, osteogenic, and adipogenic lineages. Furthermore, human cells were detected by IHC and it was observed that the iron sucrose-labeled MSCs after transplantation into lapine IVDs were traceable up to 3 months (endpoint of study). After transplantation of MSC into the lapine IVD, the total number of viable cells decreased in the lapine IVD to a similar extent, regardless if the MSCs were iron labeled or not.

The in vitro results regarding chondrogenic differentiation capability of iron sucrose-labeled human MSCs are in alignment with a previous in vitro study regarding differentiation into the chondrogenic lineage, where similar results of a low, yet detectable, ECM accumulation (day 28) and collagen II expression were observed [6].

However, in general, regarding the effects of iron labeling on differentiation of MSCs into the chondrogenic lineage, there are previously a few reports that display divergent results [34 –36]. In this study, differentiation into the osteogenic and adipogenic lineages was observed, which indicates that the stemness of the MSCs is not influenced in a high degree by the iron sucrose-labeling procedure. Furthermore, in this study, the iron-labeled cells displayed a strong signal (blue color detected by iron staining) and were traceable up to 3 months after transplantation in the lapine IVDs (ex vivo). The obtained results are comparable with previous studies of migrating cells using other iron-labeling techniques, for example, iron nanoparticles (SPIOS), for labeling of MSCs in in vitro and in vivo [8 –10,37,38]. Currently, other iron-labeling compounds, for example, ferumoxytol–heparin–protamine complex, are under investigation in animal models as a cell tracer and have shown preliminary good outcome on, for example, detection of labeled cells with MRI methodology [39]. Recently, cellular migration has been monitored in cartilaginous tissues, for example, knee joint using fluorochrome-labeling cell tracers (arthritis murine model) [40] and using SPIOS (Endorem, no longer commercial available) in normal knee joints and IVDs (lapine model) [38]. However, in general, the disadvantages of fluorochrome-based cell tracers are that these maintain their signal only for a shorter time period and are not approved for clinical use. In comparison, iron sucrose (Venofer) has a known and favorable risk profile (includes side effects as allergic reactions) in humans.

Regarding the cell viability measurements performed in this study, the investigated cells were isolated from the transplanted lapine IVDs and consisted of a mixture of injected human MSCs and the original local resident lapine IVD cells.

At the time point of 1 month after transplantation, the cell viability analyses displayed a similar and good survival rate of cells isolated from transplanted IVDs, compared to the cells isolated from the noniron-labeled MSCs injected with IVDs and cells from the undisturbed naive IVD. At the time point of 3 months after transplantation, about a 25% decrease in cell survival of the cells within the IVDs (both for iron-labeled and noniron-labeled MSC-injected IVDs) was observed compared to the naive undisturbed control IVD. Hence, no major differences in terms of cell survival were observed between the iron-labeled or noniron-labeled MSCs. In histologies, stained for glycosaminoglycans, no major differences were observed in ECM accumulation in IVDs transplanted with iron-labeled or noniron-labeled samples compared to the naive IVDs.

Presumably, at this time point, this decrease in the survival rate of the transplanted MSCs is to be expected and may be considered as a normal response to the new localization and exposure of environmental factors for the transplanted human MSCs within the lapine IVD and/or a response to the transplantation procedures.

Limitations of this study were that a more distinct differentiation of the human MSCs into the chondrogenic lineage in the iron-labeled cell pellets may have been observed in the in vitro studies, if the cell pellet cultures would have been maintained for a longer time period. Furthermore, which cell type (naive or transplanted cells) that accounted for the decrease of cell viability after cell transplantation to the IVD could not be determined in the present study.

Conclusions

The results from this study point in the direction that iron sucrose (Venofer) (which is currently in clinical use in other applications) can be used as a nontoxic in vivo cell tracer in transplantation of MSCs. For example, labeling MSCs with iron sucrose could be performed in experimental designs where tissue samples containing iron-labeled cells can be detected by histological examination. The investigated iron sucrose compound provides a stable detectable signal and was found in all the investigated animals up to 3 months after transplantation (endpoint of study). Furthermore, in the in vitro analyses it was observed that the viability of iron-labeled MSCs was not affected, while the differentiation into the chondrogenic lineage of the MSCs was somewhat influenced in the iron-labeled cell pellets compared to the controls. The iron labeling of MSCs appears to slightly delay the differentiation of these cells into the chondrogenic lineage over time. However, considering that the iron-labeled MSCs were able to form cell pellets, this indicates a sufficient chondrogenic differentiation capacity.

The results from this study demonstrate that iron labeling of human MSCs with iron sucrose can be used for monitoring of cellular distribution after stem cell transplantations and hence provide important knowledge when exploring new different cell therapy applications, for example, degenerated cartilaginous tissues.

Footnotes

Acknowledgments

This study was supported with grants from the Swedish Research Council, the Dr. Felix Neubergh Foundation, ALF agreement Vastra Gotaland, the Hjalmar Svensson Foundation, Sahlgrenska University hospital funds, VINNOVA, Vilhelm & Martina's science fund, and BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden.

Author Disclosure Statement

No competing financial conflicts exist.

References

Horwitz

, Gordon

, Koo

, Marx

, Neel

, McNall

, Muul

and Hofmann

. (2002). Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci U S A, 99:8932–8937.

Bobis

, Jarocha

and Majka

. (2006). Mesenchymal stem cells: characteristics and clinical applications. Folia Histochem Cytobiol, 44:215–230.

Barry

. (2003). Biology and clinical applications of mesenchymal stem cells. Birth Defects Res C Embryo Today, 69:250–256.

Abdallah

and Kassem

. (2008). Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther, 15:109–116.

Svenberg

, Remberger

, Svennilson

, Mattsson

, Leblanc

, Gustafsson

, Aschan

, Barkholt

, Winiarski

, Ljungman

and Ringden

. (2004). Allogenic stem cell transplantation for nonmalignant disorders using matched unrelated donors. Biol Blood Marrow Transplant, 10:877–882.

Papadimitriou

, Thorfve

, Brantsing

, Junevik

, Baranto

and Barreto Henriksson

. (2014). Cell-viability and chondrogenic differentiation capability of human mesenchymal stem cells after iron-labelling with iron sucrose. Stem Cells Dev, 23:2568–2580.

Grootendorst

, Jose

, Fratila

, Visscher

, Velders

, Ten Haken

, Van Leeuwen

, Steenbergen

, Manohar

and Ruers

. (2013). Evaluation of superparamagnetic iron oxide nanoparticles (Endorem(R)) as a photoacoustic contrast agent for intra-operative nodal staging. Contrast Media Mol Imaging, 8:83–91.

Wimpenny

, Markides

and El Haj

. (2012). Orthopaedic applications of nanoparticle-based stem cell therapies. Stem Cell Res Ther, 3:13

Svanvik

, Henriksson

, Karlsson

, Hagman

, Lindahl

and Brisby

. (2010). Human disk cells from degenerated disks and mesenchymal stem cells in co-culture result in increased matrix production. Cells Tissues Organs, 191:2–11.

10.

Liu

and Frank

. (2009). Detection and quantification of magnetically labeled cells by cellular MRI. Eur J Radiol, 70:258–264.

11.

El Haj

, Glossop

, Sura

, Lees

, Hu

, Wolbank

, van Griensven

, Redl

and Dobson

. (2015). An in vitro model of mesenchymal stem cell targeting using magnetic particle labelling. J Tissue Eng Regen Med, 9:724–733.

12.

Handley

, Goldschlager

, Oehme

, Ghosh

and Jenkin

. (2015). Mesenchymal stem cell tracking in the intervertebral disc. World J Stem Cells, 7:65–74.

13.

, Feng

, Huang

and Yan

. (2012). The application of super paramagnetic iron oxide-labeled mesenchymal stem cells in cell-based therapy. Mol Biol Rep, 40:2733–2740.

14.

Markides

, Kehoe

, Morris

and El Haj

. (2013). Whole body tracking of superparamagnetic iron oxide nanoparticle-labelled cells—a rheumatoid arthritis mouse model. Stem Cell Res Ther, 4:126

15.

Knoblich

. (2008). Mechanisms of asymmetric stem cell division. Cell, 132:583–597.

16.

Mukherjee

, Kong

and Brat

. (2014). Cancer stem cell division: when the rules of asymmetry are broken. Stem Cells Dev, 24:405–416.

17.

Singh

, Reed

, Noble

, Cangiano

and Van Wyck

. (2006). Effect of intravenous iron sucrose in peritoneal dialysis patients who receive erythropoiesis-stimulating agents for anemia: a randomized, controlled trial. Clin J Am Soc Nephrol, 1:475–482.

18.

Kosch

, Bahner

, Bettger

, Matzkies

, Teschner

and Schaefer

. (2001). A randomized, controlled parallel-group trial on efficacy and safety of iron sucrose (Venofer) vs iron gluconate (Ferrlecit) in haemodialysis patients treated with rHuEpo. Nephrol Dial Transplant, 16:1239–1244.

19.

Barot

, Parejiya

, Mehta

, Shelat

and Shah

. (2013). Physicochemical and structural characterization of iron-sucrose formulations: a comparative study. Pharm Dev Technol, 19:513–520.

20.

Lawen

and Lane

. (2013). Mammalian iron homeostasis in health and disease: uptake, storage, transport, and molecular mechanisms of action. Antioxid Redox Signal, 18:2473–2507.

21.

Wagner

, Saffrich

and Ho

. (2008). The stromal activity of mesenchymal stromal cells. Transfus Med Hemother, 35:185–193.

22.

Dominici

, Le Blanc

, Mueller

, Slaper-Cortenbach

, Marini

, Krause

, Deans

, Keating

, Prockop

and Horwitz

. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8:315–317.

23.

Horwitz

, Le Blanc

, Dominici

, Mueller

, Slaper-Cortenbach

, Marini

, Deans

, Krause

and Keating

. (2005). Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy, 7:393–395.

24.

Karlsson

, Brantsing

, Svensson

, Brisby

, Asp

, Tallheden

and Lindahl

. (2007). Differentiation of human mesenchymal stem cells and articular chondrocytes: analysis of chondrogenic potential and expression pattern of differentiation-related transcription factors. J Orthop Res, 25:152–163.

25.

Delorme

and Charbord

. (2007). Culture and characterization of human bone marrow mesenchymal stem cells. Methods Mol Med, 140:67–81.

26.

Kuroda

and Dezawa

. (2014). Mesenchymal stem cells and their subpopulation, pluripotent muse cells, in basic research and regenerative medicine. Anat Rec (Hoboken), 297:98–110.

27.

Johnstone

, Hering

, Caplan

, Goldberg

and Yoo

. (1998). In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res, 238:265–272.

28.

Fowler

and Greenspan

. (1985). Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O. J Histochem Cytochem, 33:833–836.

29.

Stigen

and Kolbjornsen

. (2007). Calcification of intervertebral discs in the dachshund: a radiographic and histopathologic study of 20 dogs. Acta Vet Scand, 49:39

30.

Hansen

and Weinfeld

. (1959). Hemosiderin estimations and sideroblast counts in the differential diagnosis of iron deficiency and other anemias. Acta Med Scand, 165:333–356.

31.

Moewis

,. (1978). Histopatologisk Teknik. Almqvist & Wiksell, Uppsala, p. 201.

32.

Scott

, Quintarelli

and Dellovo

. (1964). The chemical and histochemical properties of Alcian Blue. I. The mechanism of Alcian Blue staining. Histochemie, 4:73–85.

33.

Goldstein

and Horobin

. (1974). Surface staining of cartilage by Alcian blue, with reference to the role of microscopic dye aggregates in histological staining. Histochem J, 6:175–184.

34.

Kostura

, Kraitchman

, Mackay

, Pittenger

and Bulte

. (2004). Feridex labeling of mesenchymal stem cells inhibits chondrogenesis but not adipogenesis or osteogenesis. NMR Biomed, 17:513–517.

35.

Heymer

, Haddad

, Weber

, Gbureck

, Jakob

, Eulert

and Noth

. (2008). Iron oxide labelling of human mesenchymal stem cells in collagen hydrogels for articular cartilage repair. Biomaterials, 29:1473–1483.

36.

Farrell

, Wielopolski

, Pavljasevic

, van Tiel

, Jahr

, Verhaar

, Weinans

, Krestin

, O'Brien

, van Osch

and Bernsen

. (2008). Effects of iron oxide incorporation for long term cell tracking on MSC differentiation in vitro and in vivo. Biochem Biophys Res Commun, 369:1076–1081.

37.

Jing

, Yang

, Duan

, Xie

, Chen

, Li

and Tan

. (2008). In vivo MR imaging tracking of magnetic iron oxide nanoparticle labeled, engineered, autologous bone marrow mesenchymal stem cells following intra-articular injection. Joint Bone Spine, 75:432–438.

38.

Barreto Henriksson

, Lindahl

, Skioldebrand

, Junevik

, Tangemo

, Mattsson

and Brisby

. (2013). Similar cellular migration patterns from niches in intervertebral disc and in knee joint regions detected by in situ labeling: an experimental study in the New Zealand white rabbit. Stem Cell Res Ther, 4:104

39.

Thu

, Bryant

, Coppola

, Jordan

, Budde

, Lewis

, Chaudhry

, Ren

, Varma

, Arbab

and Frank

. (2012). Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by magnetic resonance imaging. Nat Med, 18:463–467.

40.

Koelling

, Kruegel

, Irmer

, Path

, Sadowski

, Miro

and Miosge

. (2009). Migratory chondrogenic progenitor cells from repair tissue during the later stages of human osteoarthritis. Cell Stem Cell, 4:324–335.