Mesenchymal Stem Cells Support Neuronal Fiber Growth in an Organotypic Brain Slice Co-Culture Model

Abstract

Mesenchymal stem cells (MSCs) have been identified as promising candidates for neuroregenerative cell therapies. However, the impact of different isolation procedures on the functional and regenerative characteristics of MSC populations has not been studied thoroughly. To quantify these differences, we directly compared classically isolated bulk bone marrow-derived MSCs (bulk BM-MSCs) to the subpopulation Sca-1⁺Lin⁻CD45⁻-derived MSCs⁻ (SL45-MSCs), isolated by fluorescence-activated cell sorting from bulk BM-cell suspensions. Both populations were analyzed with respect to functional readouts, that are, frequency of fibroblast colony forming units (CFU-f), general morphology, and expression of stem cell markers. The SL45-MSC population is characterized by greater morphological homogeneity, higher CFU-f frequency, and significantly increased nestin expression compared with bulk BM-MSCs. We further quantified the potential of both cell populations to enhance neuronal fiber growth, using an ex vivo model of organotypic brain slice co-cultures of the mesocortical dopaminergic projection system. The MSC populations were cultivated underneath the slice co-cultures without direct contact using a transwell system. After cultivation, the fiber density in the border region between the two brain slices was quantified. While both populations significantly enhanced fiber outgrowth as compared with controls, purified SL45-MSCs stimulated fiber growth to a larger degree. Subsequently, we analyzed the expression of different growth factors in both cell populations. The results show a significantly higher expression of brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor in the SL45-MSCs population. Altogether, we conclude that MSC preparations enriched for primary MSCs promote neuronal regeneration and axonal regrowth, more effectively than bulk BM-MSCs, an effect that may be mediated by a higher BDNF secretion.

Introduction

Studying neuronal regeneration and regrowth is important in potentially treating neurological diseases associated with the loss of neurons and their corresponding fiber projections. In this context, stem cells that positively influence neuronal regeneration and axonal regrowth are of great interest for the development of cell therapeutic strategies.

Mesenchymal stem cells (MSCs) are multipotent precursors, which are capable of differentiation into bone, cartilage, and adipose tissues. They can be obtained easily from, for example, adult bone marrow (BM), adipose tissue, or umbilical cord blood. Conventionally, MSCs are isolated from human or animal tissue through plastic adhesion and culture procedures [1 –3].

Recent reports and reviews suggest a beneficial role of MSCs in recovery after neuronal injury and in neurodegenerative diseases like Parkinson's disease [4 –8]. For example, positive effects of MSCs on fiber regeneration have been shown under various experimental conditions, such as cell cultures [9,10], organotypic slice culture models [11 –13] and also in vivo [14,15]. Besides the synthesis of extracellular matrix molecules, stabilization of the microenvironment, and immune modulatory properties, the production of humoral factors appears to be one of the relevant processes for the observed regenerative effects of MSCs [16,17]. In this context, the expression and secretion of growth factors such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), or basic fibroblast growth factor (bFGF, also known as FGF2) have been elucidated [9,13,14,18 –20].

MSCs are intrinsically heterogeneous and are comprised of a diverse repertoire of distinct subpopulations (for review see Phinney [21]). It is accepted that methods used to isolate, expand, and cultivate MSCs significantly affect their overall composition and therefore their therapeutic potential [22 –25]. Current limitations in the use of MSCs underline the need to identify specific surface markers that can be used to investigate the physiological functions and biological properties of MSCs [26]. Morikawa et al. have recently shown that MSCs need not necessarily be isolated by culture expansion, but can be prospectively isolated from primary tissue, yielding a highly enriched homogeneous subpopulation [3]. These PαS cells are a subpopulation of the very small embryonic-like stem cell population described earlier by Kucia et al. that have shown a broad differentiation potential beyond the mesenchymal lineage in vitro [27,28]. The prospective isolation of MSCs will certainly have to be considered for safer and more effective clinical treatments in the future [26]. Therefore, gaining knowledge about the different MSC populations and the use of standardized protocols for the preparation of homogenous cell populations and their expansion is integral.

We set out to investigate the impact of the isolation procedure on the functional and regenerative characteristics of MSC populations. In this study, bulk BM-derived MSCs (bulk BM-MSCs), directly expanded from cell suspensions of isolated BM of 8-week-old mice, were compared to Sca-1⁺Lin⁻CD45⁻-derived MSCs (SL45-MSCs), which were sorted from lineage depleted BM suspensions via fluorescence-activated cell sorting (FACS) from mice of the same age. The properties of both populations were analyzed in regards to their (i) ability to form fibroblast colony-forming units (CFU-f), (ii) general appearance and morphology, (iii) expression of stem cell markers and growth factors, and (iv) neuronal fiber growth promoting potential. To examine fiber growth, an organotypic brain slice co-culture model of the mesocortical dopaminergic projection system consisting of brain slices of the ventral tegmental area/substantia nigra (VTA/SN) and the prefrontal cortex (PFC) of newborn mice was employed. Fiber density in the border region between the two slices was quantified after co-cultivation with either BM-MSCs or sorted SL45-MSCs underneath the slice co-cultures using biocytin tracing and subsequent automated image analysis. The growth factor BDNF was applied to the co-culture medium as a positive control for fiber growth enhancement. The co-cultures serve as a model of both development and regeneration, as the growth of fiber connections under ex vivo conditions strongly correlates with the development of neuronal circuits in vivo, and the dopaminergic projections in the neonatal mouse brains are cut during preparation. Therefore, this model is suitable to analyze growth promoting effects of substances and cells in a context that is close to the in vivo situation, as demonstrated in a number of previous studies [29 –31].

Materials and Methods

Animals

For the preparation of the organotypic brain slice co-cultures, neonatal mouse pups of postnatal day 2 (P2) were used (C57BL/6, own breed; animal house of the Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany). For the isolation of the MSC populations, 8-week-old C57BL/6 mice were used (Medizinisch-Experimentelles Zentrum, University of Leipzig, Leipzig, Germany). Both genders were used for the preparation of the organotypic brain slice co-cultures and the isolation of both MSC populations. No gender-specific differences between male or female mice were observed. The animals were housed under standard laboratory conditions, under a 12 h light–12 h dark cycle and allowed access to lab food and water ad libitum.

All of the animal use procedures were approved by the Committee of Animal Care and Use of the relevant local governmental body in accordance with the law of experimental animal protection.

Isolation and expansion of investigated cells

Isolation of murine BM

BM was isolated following a method modified from Morikawa et al. [3]. Tibiae and femura were dissected out of 8±1-week-old C57BL/6 mice. After cleaning, the bones were crushed with a pestle, and marrow was flushed out with 5 mL of wash buffer [phosphate buffered saline (PBS) with 0.3% citrate phosphate dextrose buffer (both Sigma-Aldrich, Taufkirchen, Germany) and 1% bovine serum albumin (BSA; SERVA, Heidelberg, Germany)]. The resulting solution was passed through a 0.45 μm cell strainer (Greiner Bio-One GmbH, Frickenhausen, Germany) before proceeding. The remaining bone chips were further minced using scissors and digested at 37°C using an enzyme solution: Accutase containing penicillin/streptomycin (Pen/Strep 100 U/mL and 100 μg/mL, respectively; both PAA Laboratories GmbH, Cölbe, Germany), 2 mM calcium chloride (Sigma-Aldrich), and 1 mg/mL collagenase IV (SERVA). Afterward, the pooled cell fractions were then washed once with wash buffer and manually counted using a Neubauer counting chamber (Carl Roth GmbH andCo. KG, Karlsruhe, Germany).

Derivation of MSCs from murine BM

About 10⁷ bulk BM cells were seeded in 75 cm² culture flasks (Greiner Bio-One GmbH) and cultivated in bulk BM-MSCs maintenance medium [alpha-modification of Eagle's minimal essential medium with 10% fetal bovine serum (FBS), supplemented with Pen/Strep (100 U/mL and 100 μg/mL, respectively; all from PAA Laboratories)] to derive MSCs. After 2 days, an initial washing step with PBS was performed to remove nonadherent cells. Afterward, fresh bulk BM-MSCs maintenance medium was applied. For further expansion the medium was changed every 2–3 days. Cells were passaged via trypsinization (using trypsin; PAA Laboratories) when reaching 80% confluency. Passages 3–5 have been used for all experiments in this study except for the colony forming unit-fibroblast (CFU-f) assay (see below).

Isolation of SL45-MSCs

The isolation procedure was adapted from Kucia et al. and Morikawa et al. [3,28]. Mature blood cells were removed from BM preparations via the magnetic activated cell sorting lineage depletion kit (biotinylated antibody cocktail; Miltenyi Biotech, Bergisch Gladbach, Germany). Lineage negative (Lin⁻) cells were stained with anti-Sca-1-phycoerythrin(-PE) and anti-CD45-PE-carbocyanine(-Cy)7 in addition to Streptavidin-Alexa-Fluor-488 to label and exclude any remaining Lin⁺ cells (all from eBiosciences, Frankfurt a.M., Germany), for 30 min at 4°C using saturation concentrations recommended by the manufacturer. To discriminate living cells from dead cells and debris, Hoechst (5 μg/mL) and propidium iodide (PI; 2 μg/mL) stains (all Sigma-Aldrich) were employed. Sca-1⁺CD45⁻Lin⁻Hoe⁺PI⁻ (SL45)-MSCs were sorted by FACS in FACS sort buffer (PBS with 1% FBS, 1 mM ethylenediaminetetraaceticacid, and 25 mM HEPES; all Sigma-Aldrich; 0.2 μm sterile filtered) using a BD FACS Vantage SE cell sorter (BD Biosciences, Heidelberg, Germany).

Cultivation of SL45-MSCs

Sorted SL45-MSCs were cultivated in 25 cm² culture flasks (Greiner Bio-One GmbH) in bulk BM-MSCs maintenance medium until passage 2. Afterward, cells were cultivated in SL45-MSCs maintenance medium [alpha MEM with 30% FBS, Pen/Strep (100 U/mL and 100 μg/mL, respectively); PAA Laboratories] and 50 ng/mL recombinant human FGF2 (Peprotech, Hamburg, Germany). The administration of FGF2 and additional serum to the medium was needed for the prolonged in vitro growth of SL45-MSCs, as the cells would stop growing after 3 passages without additional FGF2. For further expansion, the medium was changed every 2–3 days. Upon reaching 80% confluence the cells were passaged using Accutase (PAA Laboratories). Passages 3–5 have been used for all experiments in this study except for the CFU-f assay (see below).

CFU-f assay

To empirically determine the frequency of CFU-f in cell suspensions, freshly isolated cells, either sorted SL45-MSCs (starting with 1,000 cells) or bulk BM-MSCs (starting with 10⁷ cells), were cultivated for 14 days in Ø 3 cm culture dishes or in 75 cm² culture flasks, respectively (both Greiner Bio-One GmbH). The cell numbers are a consequence of intrinsic differences in availability of the cells and their respective colony frequencies. The frequency of colony forming cells in bulk BM-MSCs is far lower than in the SL45 population. Plating the same number of cells would therefore generate either too many SL45-derived colonies or too few BM-MSC-derived colonies for reliable quantification. For this reason, we adjusted the input cell number to yield quantifiable numbers of colonies.

The CFU-f assays were performed for both MSC populations in bulk BM-MSC maintenance medium. The cells were fixed using methanol (AppliChem GmbH, Darmstadt, Germany) and stained using Giemsa stain (Sigma-Aldrich). The resulting primary colonies were counted using a binocular microscope.

Immunofluorescence staining

The two MSC preparations from either bulk BM or SL45 cells were characterized by applying immunofluorescence staining after cultivation under maintenance conditions. When the cultures reached 80% confluency, cells were fixed for 10 min with 4% paraformaldehyde (PFA; Merck, Darmstadt, Germany) in PBS at room temperature (RT). After the fixation step, cells were washed with PBS with 0.5% Tween 20 (PBS-T; both Sigma-Aldrich). Reactive PFA endings were saturated in a 30 min incubation step with 20 mM glycine (Carl Roth GmbH and Co. KG) in PBS. After permeabilization with 0.1% Triton-X 100 (Applichem GmbH) for 5 min, cells were washed and incubated over night at 4°C in blocking solution [PBS-T with addition of 3% FBS (PAA Laboratories), 1% BSA (SERVA)] and primary antibodies: mouse anti-βIII tubulin (1:500; Promega, Mannheim, Germany) and rabbit anti-nestin (1:400; Chemicon, Temecula, CA). Cells were washed again and stained for 1.5 h at RT in blocking solution containing secondary antibodies: donkey anti-mouse and donkey anti-rabbit conjugated with Cy2 or Cy3 (1:400/1:800, respectively; both Jackson Immunoresearch, West Grove, PA) followed by washes with Tris-buffered saline with 0.5% Tween 20 (Sigma-Aldrich). Finally, staining of the cell nuclei was performed using Hoechst 33342 (Sigma-Aldrich) at concentration of 5 μg/mL for 10 min. Then cells were washed again, embedded in fluorescence mounting medium (Dako Deutschland GmbH, Hamburg, Germany), and immunofluorescence labeling was analyzed using an inverted microscope (Nikon Eclipse Ti) equipped for the detection of Cy2, Cy3 and Hoechst.

RNA extraction and quantitative real-time polymerase chain reaction

Quantitative real-time polymerase chain reaction (qPCR) was conducted to quantify the expression of (i) stem cell markers nestin and βIII tubulin under maintenance conditions and (ii) growth factors: BDNF, ciliary neurotrophic factor (CNTF), FGF2, GDNF, hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF1), NGF, and neuregulin 1 (NRG1) after cultivation in maintenance medium or in brain slice culture medium (for composition see section “Preparation and cultivation of slice co-cultures”) in both bulk BM-MSCs and SL45-MSCs.

For gene expression analysis under co-culture cultivation conditions (meaning in brain slice culture medium, rather than in co-culture with brain slices), 1 day after seeding of the bulk BM-MSCs or SL45-MSCs, respectively, into 25 cm² culture flasks, maintenance medium was changed to brain slice culture medium. Corresponding to the duration of cultivation of cells underneath the co-cultures, after 3.5 days cells were harvested for RNA-extraction. Total RNA was isolated using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. RNA concentration was measured with NanoDrop1000 (NanoDrop Technologies, Wilmington, DE).

Reverse transcription was performed using the RevertAidTM H Minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) with 0.1 μg total RNA and oligo(dT)18 primer in 20 μL total reaction volume. qPCR was conducted using a LightCycler (Roche Diagnostics, Mannheim, Germany) in a total volume of 10 μL per capillary [LightCycler^® Capillaries (20 μL); Roche Applied Science, Mannheim, Germany] containing 5 μL QuantiTect SYBR Green 2×Master Mix (Roche Applied Science), 0.4 μL cDNA, 1 μL primer (5 μM each) specific for the different target genes or the reference genes [mitochondrial ribosomal protein L32 (MrpL32) and ubiquitin (Ubc)], and 3.6 μL water. Sequences of all used primers are listed in the Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/scd). The Hot Start Polymerase (contained in the QuantiTect SYBR Green 2× Master Mix) was activated by a 15 min preincubation at 95°C, followed by 55 amplification cycles at 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. A melting curve analysis was performed to verify correct qPCR products. The following appropriate controls have been used: no template control (water) and “reverse transcription-minus control,” to exclude the presence of genomic DNA in the samples. Quantification of gene expression was performed by the ΔCP method with MrpL32 and Ubc serving as reference housekeeping genes.

Determination of the BDNF concentration in the conditioned media

To further investigate growth factor production and secretion on the protein level, the concentration of BDNF has been quantified in conditioned brain slice culture medium. Conditioned medium of cells that were used for RNA extraction (see above) was collected and kept at−80°C until analysis. BDNF levels were measured using a multiplexed particle-based flow cytometric cytokine assay [32]. Assay kits were purchased from Millipore (Zug, Switzerland). The procedures closely followed the manufacturer's instructions. The analysis was conducted using a conventional flow cytometer (Guava EasyCyte Plus; Millipore).

Preparation and cultivation of slice co-cultures

Slice co-cultures consisting of a prefrontal and a mesencephalic slice were prepared from P2 mice and cultivated according to the “static” culture protocol using a transwell system as described previously for rats [29,33]. After decapitation of the pups, the brains were removed from the skull and immersed into low melting agar. Forebrain- and mesencephalon-containing brain parts were separated with coronal cuts and the resulting tissue blocks were fixed onto the specimen stage of a vibratome (Leica, Typ VT 1200S, Nussloch, Germany). Coronal sections (thickness 300 μm) were cut at mesencephalic and forebrain levels to obtain slices of the VTA/SN and the PFC, respectively. A scheme to illustrate the preparation procedure is shown in Fig. 1. Four co-cultures were placed on one membrane insert (0.4 μm, Millicell-CM; Millipore) in a six-well plate (NuncTM Multidish; Thermo Scientific via VWR International GmbH, Dresden, Germany), filled with 1 mL of the brain slice culture medium [25% MEM, 25% Basal Medium Eagle without glutamine, 25% horse serum, glutamine to a final concentration of 2 mM, gentamicin 50 μg/mL (all from Invitrogen GmbH, Darmstadt, Germany), and 0.6% glucose (Hospital Pharmacy, University of Leipzig, Leipzig, Germany), pH adjusted to 7.2].

FIG. 1.

Preparation of brain slice co-cultures of the mesocortical dopaminergic projection system [(A), black arrow]. After decapitation of the mouse pups, the brain was removed from the skull under sterile conditions. Coronal cuts were made at the level of forebrain and mesencephalon [(A), dashed lines]. The brain parts were fixed on the specimen stage of a slicer and cut as indicated [(B), dashed lines] to isolate PFC (*) and VTA/SN (#), respectively. Coronal sections of 300 μm thickness were cut and resulting sections were placed as co-cultures on the moistened membranes (C) and inserted in six-well plates filled with brain slice culture medium. After DIV10 the slices were fixed and the biocytin tracing was visualized. (D) Schematic co-culture slice to illustrate the localization of the PFC, the border region and the VTA/SN in greater magnification. PFC, prefrontal cortex; VTA/SN, ventral tegmental area/substantia nigra; DIV, days in vitro.

Twenty-four hours before the preparation of the organotypic co-cultures, 10⁵ bulk BM-MSCs or SL45-MSCs, respectively were seeded on poly-l-lysine (Sigma-Aldrich)-coated glass slides (Carl Roth GmbH and Co. KG) and were cultivated in the respective maintenance medium (see above). After the completion of the preparation these glass slides were transferred underneath the membrane inserts with the organotypic co-cultures. Co-cultures were kept at 37°C and 5% CO₂. Culture medium was changed every 2–3 days and glass slides were changed at days in vitro (DIV)4 and DIV7. The cells used for the exchange were derived from continuing cultures of the original input cells, ensuring that cells of the same batch and age were used throughout each experiment. Control cultures were cultivated without any additional cells or substances. As a reference for the growth promoting potential, BDNF (75 ng/mL; Peprotech) was added to the brain slice culture medium with every medium exchange in an additional experimental group (BDNF group). For detailed information see also Fig. 2.

FIG. 2.

Time schedule indicating preparation at DIV0 and subsequent medium changes, exchange of glass slides with bulk BM-MSCs and SL45-MSCs, tracing at DIV8 and fixation at DIV10. BDNF was applied after every medium exchange to the “BDNF group.” BM-MSCs, bone marrow-derived mesenchymal stem cells; BDNF, brain-derived neurotrophic factor; SL45-MSCs, Sca-1⁺Lin⁻CD45⁻-derived MSCs.

Tracing procedure, fixation, and visualization of fibers

Biocytin tracing, fixation, and subsequent processing of the co-cultures were performed as previously described [29,31,33]. At DIV8, small biocytin crystals (Sigma-Aldrich) were placed on the VTA/SN part of the co-cultures under binocular control. After 2 h, the co-cultures were rinsed carefully with brain slice culture medium. To allow the anterograde transport of the tracer, the co-cultures were reincubated for 48 h.

At DIV10, the co-cultures were fixed in a solution consisting of 4% PFA, 0.1% glutaraldehyde (SERVA), and 15% picric acid (Sigma-Aldrich) in phosphate buffer (PB, 0.1 M, pH 7.4) for 2 h and were subsequently washed with PB. Afterward, co-cultures were cut into 50 μm thick slices with the vibratome and collected in PB.

Biocytin was visualized with the avidin-biotin-horseradish peroxidise complex (1:50, ABC-Elite Kit; Vector Laboratories, Inc., Burlingame, CA) and ammonium nickel (II) sulfate hexahydrate/Cobalt (II) chloride hexahydrate (Ni/Co; both Sigma-Aldrich) intensified 3,3′-diaminobenzidine hydrochloride (Sigma-Aldrich). Stained slices were mounted on glass slides and embedded with Entellan (Merck) when completely dried.

Quantification of fiber density

Microscopic images from the whole border region were obtained with a transmitted light bright field microscope (Axioskop 50; Zeiss, Oberkochen, Germany) equipped with a CCD camera at 40×magnification. The border region of the co-culture is the part where the two initially separated brain slices were grown together (for illustrations see also Figs. 1D and 3A, B2). Slices were used for analysis only if they fulfilled the following criteria: (i) the tracer was correctly placed on the VTA/SN, (ii) the major part of the VTA/SN was characterized by a dense network of biocytin-traced structures and (iii) no traced cell bodies had been observed in the target region PFC (previously described in more detail in [30]).

FIG. 3.

Biocytin labeling. (A) Overview of a biocytin-traced co-culture slice (after cultivation with bulk BM-MSCs). (B1–B3) Microscopic images of traced cell bodies in the VTA/SN (B3), traced fibers in the border region of the co-culture (B2) and in the PFC (B1) at higher magnification. Scale bars: (A) 500 μm, (B1–B3) 100 μm. PFC, prefrontal cortex; VTA/SN, ventral tegmental area/substantia nigra.

A tailored image analysis procedure had been designed to quantify the fiber density in an automated manner, described previously in detail [31]. After preprocessing and image binarization, the ratio of the number of foreground pixels (fibers) to the total number of pixels in the images was taken, yielding an estimate of the percentage of area occupied by fibers (fiber density) in the focal plane.

Statistics

Statistical significance of the frequency of CFU-f and of the expression level of nestin and βIII tubulin between the different cell populations was assayed by Student's t-test. Statistical analysis of the fiber growth promoting effect of the different MSC populations was performed using Kruskal–Wallis analysis of variance on Ranks followed by Dunn's test for multiple comparisons against the control group and against the BDNF group. Statistical significance of the expression of various growth factors (mRNA-level; BDNF additional protein-level) was determined by Student's t-test or Mann–Whitney Rank Sum Test, as indicated. All quantitative data have been analyzed with SigmaPlot 12 statistical analysis program, all significant differences were considered at a P-level below 0.05 (*P<0.05, **P<0.01, ***P<0.001).

Results

Characterization of bulk BM-MSCs and sorted SL45-MSCs under maintenance conditions

Primary CFU-f are 10,000-fold enriched in SL45-MSCs versus bulk BM-MSCs

While bulk BM-MSCs were directly expanded from cell suspensions of freshly isolated BM of 8-week-old mice, SL45-MSCs were sorted from lineage depleted BM suspensions via FACS-sorting from mice of the same age (for details see Fig. 4A1–A4).

FIG. 4.

Primary CFU-f and representative illustration of the isolation of SL45 cells. (A1–A4) SL45-MSCs were sorted from suspensions of murine bone marrow of 8-week-old mice. For this purpose, cells were stained with Hoechst, PI, lineage markers (Lin), and a combination of Sca-1 and CD45. (A1) All events plotted with regard to forward scatter (FSC) and Hoechst signal intensity. (A2) Histogram showing PI staining of Hoechst⁺ cells from (A1). (A3) Hoechst⁺, PI⁻ cells were stained for Lin expression. (A4) The dot plot shows Hoechst⁺, PI⁻, and Lin⁻ cells that were analyzed for expression of CD45 and Sca-1. Sca-1⁺, Hoechst⁺, PI⁻, Lin⁻, and CD45⁻ cells (SL45-MSCs) were found at a frequency of 3.75%. (B) After 2 weeks of cultivation, CFU-f contained in both bulk BM-MSCs and SL45-MSCs and both cell populations gave rise to newly formed colonies comprised of spindle-shaped, fibroblast-like cells. The CFU-f frequency was far higher in SL45-MSCs than in bulk BM-MSCs. Data are shown as box-whisker plots; SL45-MSCs: n=5, bulk BM-MSCs: n=8; Student's t-test, ***P<0.001. (C1, C2) Sorted SL45-MSCs are shown in the micrographs at day 0 (d0) and 14 (d14) after sorting. Scale bars: (C1) 100 μm, (C2) 250 μm. CFU-f, fibroblast colony forming units; PI, propidium iodide. Color images available online at www.liebertpub.com/scd

Primary sorted SL45-MSCs (1,000 cells) and bulk BM-MSCs (10⁷ cells) were subjected to a CFU-f assay. These cell numbers are a consequence of intrinsic differences in availability of the cells and their respective colony frequencies (for details see section “CFU-f assay” in “Materials and Methods”). Two weeks after inoculation, both unsorted and sorted cells gave rise to newly formed colonies comprising spindle-shaped, fibroblast-like cells. As an example, sorted SL45-MSCs are shown in Fig. 4C1 at day 0 and in Fig. 4C2 at day 14 after sorting, respectively. The frequency of CFU-f was 5.3×10⁻⁶±4.1×10⁻⁶ (n=8) in bulk BM-MSCs and 5.3×10⁻²±3.3×10⁻² (n=5) in SL45-MSCs (Fig. 4B). Primary CFU-f were 10,000-fold enriched in prospectively isolated SL45-MSCs compared to bulk BM-MSCs. This suggests that SL45-MSCs are a more homogeneous cell population not only with respect to surface marker expression but also in terms of functional properties.

Higher expression of nestin in SL45-MSCs versus bulk BM-MSC

Nestin and βIII tubulin are markers for immature neuronal cells. Nestin on its own, however, has been shown to mark precursor cells of both neuronal and mesenchymal lines [34 –36]. βIII Tubulin on the other hand is co-expressed in some mesenchymal cells together with nestin before any neuronal differentiation takes place [37]. A characterization of the nestin and βIII tubulin protein expression in both unsorted bulk BM-MSCs and sorted SL45-MSCs was performed using immunofluorescence staining with antibodies against these markers. We observed a difference in nestin and βIII tubulin expression between classically isolated MSCs and those derived from sorted SL45 cells (Fig. 5A, B). While almost all SL45-MSCs expressed nestin, the population of bulk BM-MSCs was heterogeneous and also contained many cells that did not express nestin. This observation was compatible with our general impression that bulk BM-MSCs are generally more heterogeneous, with some cells showing a classical spindle-shaped MSC morphology, others appearing broader and less well defined in shape. In contrast, in SL45-MSCs the predominant cell type shows a smaller spindle-shaped or oligopolar morphology with rather small or condensed nuclei, a clearly defined cell body, and a prominent expression of nestin. βIII Tubulin-positive cells occurred with similar frequency in both cell populations.

FIG. 5.

Nestin and βIII tubulin expression. (A, B) Immunofluorescence staining against nestin (red), βIII tubulin (green), and Hoechst (blue) of (A) unsorted bulk BM-MSCs and (B) sorted SL45-MSCs expanded nondifferentiated cells growing in maintenance medium: (A) Only some bulk BM-MSCs show immunoreactivity for nestin, whereas (B) almost all SL45-MSCs were positive for nestin. βIII Tubulin-positive cells occurred with similar frequency in both cell populations. (C, D) mRNA expression levels of nestin and βIII tubulin (Tubb3) in the two cell populations after cultivation under maintenance conditions are expressed as ΔCP (relative expression normalized to the housekeeper genes Ubc and MRPL32, y-axis). (C) Nestin expression was significantly higher in SL45-MSC samples (mean>400-fold compared to bulk BM-MSCs, P<0.05). (D) No significant difference in the expression of βIII tubulin was detected by quantification of the mRNA transcripts (P=0.63). Data are shown as mean±standard error of the mean, n>4 independent cell preparations, *P<0.05, Student's t-test. Scale bars: (A, B) 100 μm. MrpL32, mitochondrial ribosomal protein L32; n.s., not significant; Ubc, ubiquitin. Color images available online at www.liebertpub.com/scd

Furthermore, the expression of nestin and βIII tubulin in both populations was determined on a mRNA level. The qPCR-results confirmed our qualitative observations from the immunostaining experiments. Nestin was detectable in both MSC populations with a significant higher expression in the sorted SL45-MSCs (mean ΔCP=13.793) compared with the bulk BM-MSCs (mean ΔCP=0.0318) (Fig. 5C). The mRNA expression of βIII tubulin was comparable in both MSC populations. There was no significant difference in the number of βIII tubulin-transcripts between the two analyzed cell populations (Fig. 5D).

MSCs derived from enriched SL45-MSCs were more potent in stimulating fiber growth than classically isolated bulk BM-MSCs

After fixation of the dopaminergic co-cultures at DIV10 and visualization of the biocytin tracing, the target orientated fiber outgrowth of cell bodies originating from the VTA/SN could be observed. Neuronal processes sprouting from the black labeled cell bodies of the VTA/SN surmounted the border region between the initially separated slices and grew into the target region PFC. An example of a VTA/SN+PFC co-culture after cultivation with bulk BM-MSCs is shown in Fig. 3.

We compared the fiber growth promoting potential of MSCs derived from sorted SL45 cells and bulk BM-MSCs to BDNF-treated and untreated controls, respectively. Examples of biocytin traced fibers within the border region are shown for all groups in Fig. 6A. The quantification of the fiber density in the border region revealed a significant growth promoting effect for both MSC preparations. The corresponding box-whisker plots are shown in Fig. 6B. Both SL45-MSCs and bulk BM-MSCs exhibit a significantly stronger growth promoting effect compared with control. As expected, BDNF also enhanced fiber growth in comparison to untreated control conditions. However, the effect was significantly less pronounced than that of the co-cultures with SL45-MSCs, suggesting a greater neurosupportive stimulus of these mesenchymal cells.

FIG. 6.

Quantification of fiber density in the border region of organotypic brain slice co-cultures after application of different BM-derived stem cell populations. (A) Microscopic images of traced fibers in the border regions of the co-cultures after cultivation with (A2) classically isolated bulk BM-MSCs, (A3) SL45-MSCs in comparison to (A4) BDNF-treatment and to (A1) control conditions. (B) Fiber quantification revealed that SL45-MSCs showed the highest benefit for fiber growth, followed by bulk BM-MSCs. The effect of BDNF was less pronounced than after co-cultivation with any of the MSC preparations. Data are shown as box-whisker plots and represent the empirical distribution of fiber densities of the investigated groups, control: n=50 images, bulk BM-MSCs: n=111 images, SL45-MSCs: n=186 images, BDNF: n=93 images; **P<0.01 in comparison to control, ^# P<0.01 in comparison to BDNF, Kruskal–Wallis analysis of variance on Ranks followed by Dunn's test for multiple comparisons. Scale bar: (A1–A4) 50 μm.

Sorted SL45-MSCs expressed significantly higher levels of the growth factors BDNF and FGF2

We assessed the expression of growth factors that could be responsible for the observed fiber growth promoting effect of bulk BM-MSCs and sorted SL45-MSCs. The mRNA of the growth factors BDNF, CNTF, FGF2, GDNF, HGF, IGF1, NGF, and NRG1 was detected by qPCR in cell samples both after cultivation in maintenance medium and in brain slice culture medium, respectively. Under maintenance conditions, the mRNA of BDNF, FGF2, and NGF was significantly higher expressed in SL45-MSCs than in bulk BM-MSCs (Student's t-test, P<0.05, data not shown). Consistent with this observation, BDNF and FGF2 were expressed to a significantly greater extent in the SL45-MSC population (Fig. 7A, B) after cultivation in brain slice culture medium. Under this condition, the median ΔCP for NGF transcripts was also higher in SL45-MSCs, but the difference between the two groups was not significant (Fig. 7C).

FIG. 7.

The expression of BDNF, FGF2, and NGF in bulk BM-MSCs and sorted SL45-MSCs, quantified by quantitative real-time polymerase chain reaction. (A–C) Expression levels of BDNF, FGF2, and NGF in the two cell populations are expressed as ΔCP (relative expression normalized to the housekeeper genes Ubc and Mrpl32, y-axis). Significantly higher expression levels of (A) BDNF and (B) FGF2 were revealed in the SL45-MSC population. No significant difference in the expression of (C) NGF between the two cell populations was detected, but there is a tendency to higher expression in the SL45-MSC samples. Data are shown as box-whisker plots, n>4 independent cell preparations, *P<0.05, **P<0.01, Mann–Whitney Rank Sum Test. FGF2, basic fibroblast growth factor; NGF, nerve growth factor; n.s., not significant.

Additionally, secreted BDNF protein has been quantified in the conditioned brain slice culture medium of both cell populations. The BDNF concentration in SL45-supernatants was significantly higher (median value: 0.487 fg/mL/cell) than in bulk BM-MSC supernatants (median value: 0.026 fg/mL/cell), P<0.01, n=6. However, the resulting BDNF concentrations in the incubation media with SL45 or bulk BM-MSCs (mean values: 288 and 17 pg/mL, respectively) were lower than in the BDNF-positive control, where 75 ng/mL BDNF was added.

Taken together, these measurements show a production of important growth factors by both cells types. This might be one of the major reasons for the observed effect on fiber growth in those cultures, which could explain the additional cell-specific benefit of the MSCs over the BDNF-treated control.

Discussion

The present study confirms the previously reported neurosupportive and regenerative features of MSCs, and additionally highlights the importance of isolation and expansion conditions. We provided evidence that sorted SL45-MSCs differ from bulk BM-MSCs in certain aspects: SL45-MSCs were enriched for primary CFU-f, were more homogeneous, expressed higher protein- and mRNA-levels of nestin, and expressed higher amounts of the growth factors BDNF and FGF2 compared with the more heterogeneous bulk BM-derived cell population. Moreover, utilizing organotypic dopaminergic slice co-cultures, we have shown that both BM-MSC populations significantly enhanced neuronal fiber growth in comparison with control conditions. The growth promoting effect of the cells was more pronounced than the effect of the growth factor BDNF. Interestingly, sorted SL45-MSCs were more potent in stimulating fiber growth in our co-culture system, indicating that this cell population is enriched for cells that are responsible for the observed regenerative and trophic effects.

Conventionally cultured MSCs yield heterogeneous populations that often contain contaminating cells due to the significant variability in the isolation method, the tissue origin, and the lack of specific MSC markers [21,23,26]. Moreover, experimental artifacts introduced by inconsistent cell culture protocols or following extensive culture manipulation may result in changes of the MSCs' properties or in the aging processes of the cells, both causing complications in the utilization of MSCs as therapeutic tools (for review see Wang et al. [25]). As an example, the number of passages was shown to greatly influence the survival rate, migration, and neuroprotective capacities of MSCs [38]. With regard to the use of MSCs in clinical medicine, it is anticipated that more homogeneous cell preparations will yield more consistent clinical outcomes that exhibit both dose responsiveness and more reproducible outcomes [21]. Collectively, these studies underline the importance of preparing adequate homogenous cell populations of MSCs.

In our study, we found a higher frequency of nestin-positive cells and significantly more mRNA transcripts of nestin in SL45-MSCs compared with classically isolated bulk BM-MSCs. This is in accordance with results of other studies, where the heterogeneity of primary MSC cultures and an increase in the percentage of nestin-positive MSCs as a function of the number of passages were observed [39]. This further implicated the superior proliferative capacity of nestin-positive MSCs.

The expression of nestin and βIII tubulin in undifferentiated cells has been reported previously and has been taken as evidence for proliferative and progenitor potential [35,40 –42]. Furthermore, nestin has been described as a marker for MSCs in the recent literature [34,37,43]. In this context, it was shown that nestin-positive MSCs contain all the CFU-f activity of the BM, can self-renew in serial transplantation, and are able to differentiate toward mesenchymal lineages [34,37]. Indeed, we could confirm multilineage differentiation capacity into adipogenic, chondrogenic, and osteogenic lineages in both bulk BM-MSCs and SL45-MSCs (Supplementary Fig. S1).

Several findings point to an active role of MSCs in experimental in vitro and in vivo models of different diseases of the CNS, including Parkinson's disease, stroke, multiple sclerosis, traumatic brain or spinal cord injury [8,44 –48]. As an example, animal studies in traumatic brain injury have shown improved functional outcome when MSCs were injected intracerebrally or intravenously [49,50]. Human MSCs were beneficial after spinal cord injury due to the increase in axonal sprouting and in the number of surviving rat cortical neurons in vivo [51]. Further underlining the importance of MSCs, there are 77 clinical trials registered using MSCs in neuropathological conditions (www.clinicaltrials.gov, website of the National Institutes of Health, Bethesda, MD, search for “mesenchymal stem cells and CNS,” October 2014, eg, regarding Parkinson's disease: NCT01446614).

The regeneration of injured neuronal cells and the corresponding fiber projections are of great interest with respect to the described neurological diseases. Organotypic brain slices that can be maintained in culture for several weeks facilitate the study of cellular interactions and mechanisms in this context. Furthermore, this technique represents a powerful tool for the development of ex vivo models of neurological disorders and to investigate approaches for potential stem cell therapies (for review see Daviaud et al. [52]). For example, MSCs revealed a significant neuroprotective effect in ischemia models of organotypic hippocampal slices [19,53]. In organotypic spinal cord slices, topically applied MSCs survived, migrated into the slice, and promoted host neurite extension [11,12] and axonal outgrowth from the cortex to the spinal cord [13]. Here, we describe a beneficial effect of BM-MSC preparations with a particular focus on the clinically relevant parameter of axonal outgrowth in the dopaminergic system. Our data indicate that SL45-MSCs are more potent than bulk BM-MSCs. Therefore, SL45-MSCs appear to be particularly well suited to promote neuro(re)generation ex vivo.

Our findings show that MSC preparations derived from a defined cell population enriched for primary MSCs are advantageous in different aspects. (i) SL45-MSCs have a higher fiber growth promoting potential compared to conventional bulk BM-MSCs. (ii) A more homogenous MSC population can be obtained much faster starting from pre-enriched CFU-f, allowing for a shorter expansion phase in vitro, and hence a lower risk of culture prone alterations or mutation accumulations on the MSC population. (iii) Due to the more homogeneous population derived from enriched primary MSCs, the risk of contamination with alloreactive immune cells might potentially be lower. Summarizing, we suggest the use of cell material that is more homogeneous and highly enriched in CFU-f activity as a starting point for cell therapeutic development. This is in line with previously published studies and their conclusions [1,3,26,28,54].

Earlier studies were based on the assumption that the neuroregenerative effects of MSCs are caused by the replacement of damaged cells by differentiation into neurons or glia [55,56]. However, the current opinion favors a model of regeneration induced by the release of soluble factors, such as trophic factors. Thus, the capacity of MSCs to actively alter the microenvironment via these factors may contribute more significantly to tissue repair than their plasticity [16,24,47]. Our findings also suggest a stimulating effect mediated via soluble secreted factors as the main reason for the observed impact on fiber growth, as the cells where plated underneath the co-cultures without direct contact to the brain slices in our study. This hypothesis is in line with findings by other groups, demonstrating the secretion of trophic factors by MSCs, including BDNF, NGF, GDNF, FGF2, and the vascular endothelial growth factor [9,18,57 –59].

We detected the mRNA of the growth factors BDNF, CNTF, FGF2, GDNF, HGF, IGF1, NGF, and NRG1 by qPCR in all assessed cell samples. The significantly higher expression of BDNF and FGF2 in the SL45-MSC population, which has exemplarily been confirmed on the protein level for BDNF, could be a reason for the more pronounced effect evoked by the sorted SL45-MSC population as compared to the classically isolated bulk BM-MSCs. This might result from the homogeneity of the populations with SL45-MSCs where the cells express similar levels of growth factors, while the more heterogeneous bulk BM-MSCs consist of a mixture of both secreting and non-secreting cells.

The observed growth promoting effect of the growth factor producing MSC populations in the dopaminergic projection system is not surprising, taking into account that previous studies have shown the beneficial effects of BDNF, GDNF, and FGF2 on mesencephalic dopaminergic neurons [60 –62]. These growth factors are considered to mediate dopaminergic neuronal survival and neuroprotection. Thus, they had become potential candidates for the therapy of Parkinson's disease [63 –66]. For example, BDNF provides support for both developing and adult dopaminergic neurons [67 –70]. So BDNF resulted in enhanced survival of tyrosine hydroxylase-positive neurons and increased growth of tyrosine hydroxylase-positive fibers into striatal tissue [71]. GDNF, which is especially important for the development of embryonic dopaminergic neurons [72], induced neuronal survival and promoted the innervation of target areas [73 –76]. FGF2 stimulated the proliferation of dopaminergic progenitor cells, promoted survival and neurite outgrowth of dopaminergic neurons, and participated in nigrostriatal pathway formation and target innervation [66,77,78].

The impact of the BDNF application to the brain slice culture medium was less pronounced than the growth facilitating properties of both MSC populations under study. This is in agreement with previous studies showing that the combined application of neurotrophins (eg, BDNF and NGF) elicited greater axon growth than treatment with each neurotrophin alone [79]. Moreover, Crigler et al. revealed that MSCs express potent neuroregulatory molecules in a restricted manner in addition to growth factors like BDNF and NGF. These growth factors may further contribute to MSC-induced effects on neuronal survival and nerve regeneration [9].

Conclusion

In conclusion, both MSC preparations exerted highly beneficial effects on fiber outgrowth in organotypic brain slices of the dopaminergic system well above the level of untreated controls, and had also a stronger effect than the positive control BDNF. Thus, MSCs appear to be very well suited to promote neuro(re)generation. The outcome was dependent on the isolation and expansion conditions and we could show that the more homogeneous sorted SL45-MSCs were more potent in stimulating fiber growth, indicating an enrichment of cells that are responsible for the observed regenerative and trophic effects. The higher expression levels of nestin, BDNF, and FGF2 in SL45-MSCs compared to classically isolated BM-MSCs may serve as an explanation for the higher potential in stimulating fiber growth as demonstrated in this study.

Footnotes

Acknowledgments

The authors thank Katrin Becker and Katrin Krause (Rudolf Boehm Institute of Pharmacology and Toxicology) as well as Gabrielle Öhme and Sabine Hecht (TRM Leipzig) for excellent technical assistance. The authors are grateful to Adrian Urwyler (Cytolab, Regensdorf, Switzerland) for performing the BDNF measurements in the conditioned media. The authors thank Jennifer Ding for providing language help. The work presented in this article was made possible by funding from the German Federal Ministry of Education and Research (BMBF 1315883).

Author Disclosure Statement

None of the authors has any disclosure to declare. No competing financial interests exist.

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