Human platelet lysate stimulated adipose stem cells exhibit strong neurotrophic potency for nerve tissue engineering applications

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Human platelet lysate stimulated adipose stem cells exhibit strong neurotrophic potency for nerve tissue engineering applications

LISCHER, Mirko, et al.

Abstract

Aim: We investigated a potential strategy involving human platelet lysate (HPL) as a media additive for enhancing the neurotrophic potency of human adipose stem cells (ASC). Materials

& methods: Dorsal root ganglion explants, ASC and Schwann cells were used for in vitro axonal outgrowth experiments. Results: Remarkably, HPL-supplemented ASC promoted robust axonal outgrowth, in other words, four-times higher than fetal bovine serum-supplemented ASC and even matched to the level of Schwann cells. Further, analysis of regime of growth medium additive supplementation revealed the critical play of HPL in dorsal root ganglion and stem cells co-culture system for mounting effective axonal growth response. Conclusion: HPL supplementation significantly improved the neurotrophic potency of ASC as evidenced by the robust axonal outgrowth; these findings hold significance for nerve tissue engineering applications.

LISCHER, Mirko, et al . Human platelet lysate stimulated adipose stem cells exhibit strong

neurotrophic potency for nerve tissue engineering applications. Regenerative medicine , 2020, vol. 15, no. 3, p. 1399-1408

DOI : 10.2217/rme-2020-0031 PMID : 32308109

Available at:

http://archive-ouverte.unige.ch/unige:155274

Disclaimer: layout of this document may differ from the published version.

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Human platelet lysate stimulated adipose stem cells exhibit strong neurotrophic potency for nerve tissue engineering applications

Mirko Lischer1,2,3 , Pietro G di Summa4, Carlo M Oranges1, Dirk J Schaefer1,5, Daniel F Kalbermatten1,2,3, Raphael Guzman5,6& Srinivas Madduri*,1,2,3,5

1Department of Plastic, Reconstructive, Aesthetic & Hand Surgery, University Hospital Basel, Spitalstrasse 21, 4031, Basel, Switzerland

2Department of Pathology, University Hospital Basel, Hebelstrasse 20, 4021, Basel, Switzerland

3Department of Biomedical Engineering, University of Basel, 4123, Allschwil, Basel, Switzerland

4Department of Plastic & Hand Surgery, University Hospital Lausanne (CHUV), 1005, Lausanne, Switzerland

5Department of Biomedicine, University of Basel, Hebelstrasse 20, 4021, Basel, Switzerland

6Department of Neurosurgery, University Hospital Basel, Spitalstrasse 21, 4031, Basel, Switzerland

*Author for correspondence: srinivas.maduri@usb.ch

Aim:We investigated a potential strategy involving human platelet lysate (HPL) as a media additive for enhancing the neurotrophic potency of human adipose stem cells (ASC). Materials & methods: Dorsal root ganglion explants, ASC and Schwann cells were used forin vitro axonal outgrowth experiments.

Results:Remarkably, HPL-supplemented ASC promoted robust axonal outgrowth, in other words, four- times higher than fetal bovine serum-supplemented ASC and even matched to the level of Schwann cells.

Further, analysis of regime of growth medium additive supplementation revealed the critical play of HPL in dorsal root ganglion and stem cells co-culture system for mounting effective axonal growth response.

Conclusion:HPL supplementation significantly improved the neurotrophic potency of ASC as evidenced by the robust axonal outgrowth; these findings hold significance for nerve tissue engineering applications.

Stimulated stem cells

Stimulated stem cells Human platelet lysate

(HPL) HPL stimulation

FBS stimulation Human adipose stem cells (ASC)

Fetal bovine serum (FBS)

Stimulated cells or/and HPL

Stimulated cel ls

or/and HPL

Investigation on axonal outgrowth in vitro

Modulation of adipose stem cells’ neurotrophic capacity

First draft submitted: 6 March 2020; Accepted for publication: 30 March 2020; Published online:

20 April 2020

Keywords: adipose stem cells • axonal regeneration • growth factors • human platelet lysate • nerve tissue engineering•neurotrophic factors•Schwann cells

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Peripheral nerve injuries lead to lifelong disabilities with a huge socioeconomic burden[1]. A retrospective study in The Netherlands involving 96 patients in the years 1990–1998 observed that only 59% of patients with ulnar or median nerve trauma returned to work within a year[2]. Considering more than 300,000 cases of peripheral nerve injury in Europe annually, the improvement of the outcome holds huge potential[3]. If possible, a tension-free end-to-end anastomosis is the standard procedure for peripheral nerve repair. However, defects are often more severe due to tissue lost by injury or debridement, and nerve gaps needs to be bridged in order to allow nerve regeneration.

The current gold standard for repairing nerve defects exceeding 5 mm gap is autograft transplantation, which leads to good results but comes with several disadvantages including donor site morbidity and modality mismatch[4,5]. Nerve allografts are highly immunogenic and would require treatment with immunosuppressive drugs[6], leading to infection risk. Therefore, the need emerged for tissue-engineered nerve grafts (TENG), which may offer the viable replacement for autologous nerve graft.

TENG should consist of bioinspired architecture such as support cells, growth factors (GFs) and guidance struc- tures, in order to match with the gold standard autograft[7,8]. Physiologically, Schwann cells (SC) play an essential role in nerve repair and axonal regeneration by secreting wide range of cytokines and cell adhesive molecules[9]. However, clinical use of SC is limited due to the problems associated with cell harvest and expansion[10]. Allogeneic SC are challenging due to the problems associated with the required immunosuppressive treatment[11]. Thus, stem cell-based therapies emerged and mesenchymal stem cells (MSC) showed potential for neurotrophic support when induced into SC-like cells (SCLC)[12,13]. Not limiting to the source of bone marrow stem cells, MSC can be easily accessed in abundant quantities from adipose tissues[14–16]. Human adipose stem cells (ASC) possess neurotrophic properties and support axonal regenerationin vitroandin vivo, although their neurotrophic potency remained inferior to SC. Furthermore, ASC proliferate rapidly, possess multi-lineage capacity and maintain mesenchymal potency even after prolonged cultures[17,18]. Thus, ASC may enable the development of effective TENG, provided that the neurotrophic potency of ASC is significantly enhanced, in other words, up to the level of SC.

The critical aspect of TENG is the need to have ASC with strong neurotrophic potency. This is partly evident by the fact that transplantation of SCLC, in other words, ASC with improved neurtrophic support, results in more effective nerve regeneration[19]. However, endogenous maintenance of neurotrophic capacity of these cells remains disputed[20]. Further on, a continuous GFs supplementation and the underlying challenges to maintain the neurotrophic capacity of ASC hampered their clinical translation. Moreover, the traditional use of fetal bovine serum (FBS) as medium supplementation implies major hurdles for clinical translation. Thus, there is a great need for new strategies involving the safe and efficacious additives for improving the neurotrophic potency of ASC. Human platelet lysate (HPL) is an US FDA-approved medium supplement, which has a higher concentration of GFs than any of the other cell culture supplements including plate-rich plasma and FBS[21–23]. Several studies reported the beneficial effects of HPL for supporting cell expansion, differentiation and tissue regeneration, compared with FBS [24]. Being a human derivate, allogeneic HPL with a pathogen free certification is currently under use for human trials[25–27].

However, the impact of HPL on the neurotrophic potency of ASC still remains to be elusive for axonal regeneration. Therefore, the present study aims to elucidate the neurotrophic capacity of ASC in response to HPL supplementationin vitro. For this, ASC were preconditioned by HPL-supplemented growth medium (GM) and the resulting cells were evaluatedin vitrofor their ability to promote axonal outgrowth in comparison to FBS-stimulated ASC and gold standard SC. Furthermore, the regime of HPL stimulation was analyzed in relation to the effective axonal growth response.

Materials & methods

Growth medium & supplements

Except otherwise specified, in the present study we used DMEM (Sigma-Aldrich, Buchs, Switzerland) supplemented with 10% FBS and 0.2% penicillin–streptomycin (P/S) or 5% HPL (PLTmax; Sigma-Aldrich), 0.2 % P/S and 2U/ml of heparin sodium salt (Sigma-Aldrich).

Experimental design

Human ASC were preconditioned for 3 days by applying HPL or FBS-supplemented GM (ASCHPL+or ASCFBS+) and resulting cells were used for the axonal outgrowth assay for 2 days under the continued culture conditions, in other words, HPL-ASCHPL+or FBS-ASCFBS+(Figure 1). Subsequently, the order of supplementation of HPL

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Stimulation of ASC preceding to axonal outgrowth assay Culture

conditions

Axonal outgrowth assay in vitro

Analysis of axonal outgrowth in vitro

ASC Seeding of DRG

Preconditioning of ASC for 3 days by supple- menting media additives (HPL or/and FBS) Seeding of DRG explants on top of ASC layer under continued or altered supplementation of media additives

0 1 2 3 4 5 Time (days)

HPL-ASCHPL+

FBS-ASCFBS+

HPL-ASCFBS+

FBS-ASCHPL+

HPL+ HPL+

HPL+ HPL+

FBS+ FBS+

FBS+ FBS+

Quantitative analysis of axonal elongation and

growth area

Figure 1. Study design.Stimulation of adipose stem cells by using human platelet lysate or fetal bovine serum as potential growth medium additives and biological assessment of stimulated stem cells for promoting axonal outgrowth.

ASC: Adipose stem cell; DRG: Dorsal root ganglion; FBS: Fetal bovine serum; HPL: Human platelet lysate.

or FBS to the GM for preconditioning and for subsequent axonal growth assay was sequentially altered. For this, in a first regime, ASC’s preconditioning by using FBS-GM preceded the axonal outgrowth assay that was supplemented with HPL-GM, in other words, HPL-ASCFBS+. In a second regime, ASC’s preconditioning by using HPL-GM preceded the axonal outgrowth assay that was supplemented with FBS-GM, in other words, FBS-ASCHPL+(Figure 1). As a control, HPL or FBS supplemented GM, in other words, HPL-GM or FBS-GM were used in addition to gold standard SC that were cultured under standard conditions. Thus the following cultures conditions were applied for the axonal outgrowth experiments in a sum: HPL-ASCHPL+, FBS-ASCFBS+, HPL-ASCFBS+, FBS-ASCHPL+, HPL-GM, FBS-GM, and SC.

Isolation & culture of adipose stem cells

Adipose tissue was obtained from a healthy single human donor undergoing elective liposuction. We obtained an informed consent from the patient prior to liposuction, in addition to the ethical permission that was granted by University Hospital Basel. The isolation of ASC was performed under sterile conditions according to a well- established protocol[28]. The fat tissue was cleaned from erythrocytes by rinsing with phosphate-buffered solution (PBS) and centrifugation, further it was minced and digested with 0.15% (w/v) Type I collagenase (Gibco Life Technologies, Basel, Switzerland, cat. no. 17100017) for 1 h at 37C and centrifuged for 5 min at 1500 rpm and 4C. The resulting pellet was resuspended in a GM in other words, DMEM (Gibco, cat. no. 41965039) supplemented with 10% FBS (PAN-Biotech, Aidenbach, Germany, EU-approved, cat. no. P40-47500) and 1%

P/S (BioConcept, Allschwil, Switzerland, cat. no. 4-01F00-H). Extracted ASC were seeded at a density of 3000 cells/cm2and cultured at 37C with 5% CO2in a humid atmosphere with GM being changed every 72 h. Cells were passaged at 90% confluence by using 0.25% Trypsin-EDTA (BioConcept, cat. no. 5-51F00- H) and resulting cells at passage 2 (P2) or 3 (P3) were used for the experiments.

Isolation & culture of SC

SC were harvested and cultured according to previously described protocols, with some modifications[29,30]. Sciatic nerve from 24–48 h old rat pups were collected aseptically and subjected to digestion with 0.1% trypsin and 0.2%

collagenase in Hank’s buffered salt solution at 37C for 60 min. Cells were dissociated by repeated trituration using

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a glass Pasteur pipette. Cells were washed twice with DMEM containing 10% FBS and cultured on poly(L-lysine)- coated tissue culture plates in DMEM containing 10% FBS, 10μM cytosine arabinoside and 0.2% P/S. Exposure to cytosine arabinoside eliminates the fast-growing fibroblasts. After 24 h, the medium was replaced by GM for SC, composed of DMEM, 10% FBS, 0.2% P/S, 4μg/ml of bovine pituitary extract, and 2μM forskolin; the cultures were then maintained at 37C for 2–3 days. At 80–90% confluency, complement-mediated lysis of contaminating fibroblasts was performed using anti-Thy-1.1 IgG to remove any remaining of these cells. Purified SC were further cultured through three to four passages and then used for the further experiments.

Isolation of chicken embryonic dorsal root ganglions

Fertilized chicken eggs were obtained from Gepro Gefl¨ugelzucht AG (Flawil, Switzerland). The eggs were shipped at ambient temperature and incubated at 37.8± 0.2C under 100% relative humidity for 9 days (E9). After incubation, the eggs were cleaned with 70% ethanol and opened under a laminar airflow cabinet to collect the embryos. Dorsal root ganglions (DRG)-explants were dissected from the lumbar part of the spine following a standard dissection protocol under a stereomicroscope[31]and resulting DRG explants were collected in GM for further experiments.

Axonal outgrowth assay

Forin vitroaxonal outgrowth experiments, DRG explants isolated from 9 days old (E9) chicken embryos were seeded at a density of one per well onto 24-well plates[31]that were preseeded with cells or/and GM as detailed earlier. As detailed earlier (Figure 1), ASC and SC were cultured at a density of 8000 cells/cm2for 3 days prior to DRG seeding for axonal outgrowth assessment. Cultures were maintained in a humid atmosphere at 37C and 5%

CO2for 2 days and images were captured at 10×magnification using a mosaic image stitching option. In total, three independent experiments resulting in a total of 12 DRG explant cultures for each experimental condition were performed.

Immunocytochemistry of DRG cultures

After 48 h, DRG cultures were observed under microscope and bright field images with a phase contrast were taken at 10×magnification using a Zeiss Axio Vert.A1 inverted fluorescent microscope (Carl Zeiss AG, Germany) and a Zeiss AxioCam MRc camera (Carl Zeiss AG). DRG explants were then fixed in 4% paraformaldehyde (PFA) at room temperature (RT) for 10 min and permeabilized and blocked in 0.01 M PBS containing 0.1% Triton X-100 and 1% BSA (i.e., dilution buffer) for 20 min at RT. For immunocytochemistry, the cultures were incubated overnight at 4C with the monoclonal mouse anti-β-tubulin III (1:1000, Sigma-Aldrich, cat. no. T8578) for axons.

The cultures were then washed in PBS and incubated with secondary antibodies sheep anti-mouse Cy3 (1:500, Sigma Aldrich, cat. no. C2181) and Hoechst 33258 nuclear staining (1:1000, Sigma Aldrich, cat. no. 94403) for 1 h at RT. Subsequently, digital images were acquired at 10×magnification (numerical aperture 0.45) by using a Nikon Eclipse Ti inverted fluorescent microscope (Nikon Eclipse Ti-E, -E/B, Nikon Corporation, Japan) and a Nikon DS-Qi2 camera (Nikon Corporation). The images were automatically stitched by the Nikon NIS-Elements AR image analysis software (NIS-Elements AR Analysis 5.11.00 64-bit, Laboratory Imaging, spol. s.r.o., Czech Republic).

Quantitative measurements of axonal outgrowth

Axonal length between the periphery of the DRG explant and tip of the regenerating axons was measured by using the Image J software (NIH, MD, USA) in eight different directions representing the 0, 45, 90, 135, 180, 225, 270, 315axis. Resulting data were used for calculating the average axonal outgrowth length. Further, axonal area was measured by using the polygon selection tool of which the area of the DRG itself was subtracted with the freehand selection tool.

Statistical analysis

Data were analyzed by one-way analysis of variance following Bonferroni procedure withpost hocmultiple compar- isons using SPSS (version 15.0; SPSS, IL, USA). Values with p<0.05 were considered significant. Our assumptions for statistical analysis include normal distribution, random and independent sampling, equal variance and accurate measurements.

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0 100 200 300 400

Axonal length (µm)

HPL-GM FBS-GM

HPL-GM FBS-GM

500

*

0.0 0.5 1.0 1.5

Axonal area (mm2)

HPL-GM FBS-GM 2.0

*

Figure 2. Human platelet lysate promotes axonal outgrowth.Axonal regeneration from dorsal root

ganglion-explantsin vitro.(A)Images of dorsal root ganglion-explant cultures treated with various growth medium supplements,(B)quantitative measurements of axonal length and(C)quantitative measurements of axonal area.

Only anti-β-tubulin staining was shown. The scale bar represents 500μm. The bars represent mean±standard deviation of n=12.

Significant differences are indicated at *p<0.05.

FBS: Fetal bovine serum; HPL: Human platelet lysate.

Results

Culture conditions using HPL-GM resulted in an important axonal outgrowth from DRG explants, in other words, axonal length (inμm) and axonal area (in μm2), which are in the range of 421±37 and 1.42±0.18 respectively (Figure 2). In contrast to HPL-GM, FBS-GM resulted only in minimal axonal outgrowth, in other words, 173±42 and 1.43±0.12 (Figure 2).

Remarkably, HPL-ASCHPL+ promoted significantly robust axonal growth, in other words, 888 ±181 that is statistically comparable to the axonal growth promoted by SC, in other words, 930± 136 (Figure 3). The increase in the axonal growth supported by HPL-ASCHPL+ is about fourfold higher than the FBS-ASCFBS+, in other words, 215±47 (Figure 3). These results are consistent with axonal area measurements, in other words, 3.3±05, 0.54±0.06 and 3.7±04, for HPL-ASCHPL+, FBS-ASCFBS+ and SC, respectively (Figure 3). These findings indicate the significant enhancement of ASC’s potency for promoting the axonal regeneration in response to HPL-continuous supplementation.

Further on, HPL-ASCFBS+promoted significant axonal outgrowth, in other words, 645±75, although a slight decline is evident in comparison to HPL-ASCHPL+, in other words, 888±181 (Figure 3&Figure 4). In contrast to HPL-ASCFBS+, FBS-ASCHPL+did not result in the significant axonal outgrowth, in other words, 362±60 and 1.16±0.16 (Figure 4). These results indicate the importance of HPL supplementation to the co-culture of the DRG and ASC in combination with ASC’s HPL-prestimulation for mounting the effective axonal growth response.

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SC HPL-ASCHPL+ FBS-ASCFBS+

0 200 400 600 800 1000

Axonal length (µm)

1200

*

*

SC HPL-ASCHPL+ FBS-ASCFBS+

FBS-ASCFBS+

0.0 0.5 1.0 1.5 3.5 4.0

Axonal area (mm2) 4.5

3.0 2.5 2.0

*

*

Figure 3. Human platelet lysate stimulated adipose stem cell promote robust axonal outgrowth.Axonal

regeneration from DRG explantsin vitro.(A)Images of DRG explant co-cultures involving various cell types, in other words, DRG co-cultures involving SC that were cultured with standard growth medium for 4 days; DRG co-cultures involving HPL-stimulated adipose stem cells (ASCHPL+) that were cultured with HPL-supplemented growth medium (HPL-ASCHPL+) for 2 days and DRG co-cultures involving FBS-stimulated adipose stem cells (ASCFBS+) that were cultured with FBS-supplemented growth medium (FBS-ASCFBS+) for 2 days.(B)Quantitative measurements of axonal length and(C)quantitative measurements of axonal area. Axonal length and axonal area resulting from 4 days co-cultures were normalized to the other cultures of 2 days. Only anti-β-tubulin staining was shown. The scale bar represents 500μm. The bars represent mean±standard deviation of n=12.

Significant differences are indicated at *p<0.05.

ASC: Adipose stem cell; DRG: Dorsal root ganglion; FBS: Fetal bovine serum; HPL: Human platelet lysate; SC: Schwann cell.

Discussion

Functional regeneration of peripheral nerves is clinically critical and challenging, consequently number of patients with functional disabilities are increasing annually. Thus, there is a great need for the development of the new therapies that may overcome the morbidities associated with the gold standard autologous nerve grafts and offer new options for off-the-shelf therapies. One of the major limitations in achieving the new therapeutics is lack of the cell types matching with natural SC properties and the GF supplementation required to support cells of tissue-engineered nerve grafts (TENG).

ASC possess neurotrophic properties, but their functional efficiency remained inferior to SC. HPL contains wide range of GFs and cytokines in high concentrations and represents a potential source of autologous therapeutic

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0 200 400 600 800 1000

Axonal length (µm) HPL-ASCHPL+ HPL-ASCFBS+ FBS-ASCHPL+

1200

HPL-ASCHPL+ HPL-ASCFBS+ FBS-ASCHPL+

*

*

0.0 0.5 2.5 3.0 3.5 4.0

Axonal area (mm2) 4.5

2.0 1.5 1.0

*

*

Figure 4. Human platelet lysate and stimulated-adipose stem cell exhibit synergistic function on axonal

outgrowth.Axonal regeneration from DRG explantsin vitro.(A)Images of DRG explant co-cultures involving various cell types, in other words, DRG co-cultures involving HPL-stimulated adipose stem cells (ASCHPL+) that were cultured with HPL-supplemented growth medium (HPL-ASCHPL+) for 2 days; DRG co-cultures involving FBS-stimulated adipose stem cells (ASCFBS+) that were cultured with HPL-supplemented growth medium (HPL-ASCFBS+) for 2 days and DRG co-cultures involving HPL-stimulated adipose stem cells (ASCHPL+) that were cultured with FBS-supplemented growth medium (FBS-ASCHPL+) for 2 days.(B)Quantitative measurements of axonal length and(C)quantitative

measurements of axonal area. Only anti-β-tubulin staining was shown. The scale bar represents 500μm. The bars represent mean±standard deviation of n=12.

Significant differences are indicated at *p<0.05.

ASC: Adipose stem cell; DRG: Dorsal root ganglion; FBS: Fetal bovine serum; HPL: Human platelet lysate; SC: Schwann cell.

substance. However, the impact of HPL on ASC’s potency remains to be detailed in the context of nerve repair and axonal regeneration. Therefore, we have evaluated the axonal growth promoting properties of ASC from chicken embryonic DRG explantsin vitroand compared the outcome measurements with the gold standard SC. DRG explant consist of sensory neurons, which is a certain limitation. Nevertheless, we expected the use of this DRG system to provide relevant information, based on our earlier studies[31], on the ability of HPL-activated ASC for promoting axonal outgrowth.

Main findings of the present study are the following: significant improvement of ASC’s neurotrophic potency in response to HPL supplementation as evidenced by the robust axonal outgrowth, in other words, four-times higher than FBS, HPL-conditioned ASC appeared to be comparable to SC in the context of axonal outgrowth, which is an important achievement and HPL supplementation to co-culture of DRG and ASC in combination with ASC’s HPL-preconditioning is crucial for mounting an effective axonal regeneration.

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ASC are widely studied for nerve regeneration bothin vitroandin vivo[28,32–34]. Although these cells exhibit neurorophic properties and showed to support axonal regeneration, at least in their undifferentiated form, remained not comparable to SC. Several efforts were made for improving the ASC’s neurotrophic function by differentiating them into SCLC. However, the topic of ASC differentiation remained inconclusive as reported by several studies and the resulting benefits are not clear. Moreover, therapeutic use of differentiated ASC is hampered by the long differentiation process, in other words, 4 weeks and xenogeneic materials involved[20,35,36], with limited clinical translation. Thus, the need for improving the ASC’s neurotrophic function is continued. In the present study, we used the cells isolated from a single human donor. In a continuation study, it would be interesting to investigate the effects of HPL stimulation on ASC obtained from multiple human donors in the context of nerve regeneration, as studies have shown that ASC from different donors possess different immune-phenotypic profiles and may display different paracrine capacity. In our experiments, heparin was supplemented only to the GM containing HPL.

However, initially we examined both culture media, in other words, FBS versus HPL, supplemented with heparin 2U/ml and found no effect either on ASC or on axonal regrowth (data is not shown). Heparin appears to inhibit the proliferation of MSC at higher concentration (>100 U/ml) and no significant effects were found at lower concentrations[37]. Thus, the concentration of heparin supplemented in our study is just appropriate.

From a physiological perspective, platelets contain a great variety of GFs, and cytokines, in other words, TGF-β, PDGF-AB, PDGF-AA, PDGF-BB, IGF-1, BDNF, EGF, VEGF and bFGF that are suitable to sustain growth of a wide range of cell types[38,39]. Moreover, the concentrations of these factors are significantly higher in HPL in comparison to FBS[40]. Thus, the enhanced neurotrophic potency of ASC could be explained by the rich variety of cytokines in combination with the GFs present in HPL. FBS contains limited range of GFs at lower concentrations.

Furthermore, FBS contains gamma globulin (i.e., antibodies) content and complement proteins, which may bind to the neuronal cells in the culture and may have the undesirable effects of lysing cells in culture and interfering with axonal extension. Thus, these inhibitory factors together with limited amount of GFs may explain the limited amount of axonal growth observed in FBS-cultures.

The factors applied for thein vitrodifferentiation of ASC into SCLC, in other words, PDGF, FGF and neuregulin are abundant in HPL which may result in the improvement of ASC’s potency for axonal growth and further, 3-day preconditioning may be needed for translation of the molecular signals mediated by the HPL-constituents and for the enhancement of the endogenous mechanisms[28,41]. Wanget al.very recently reported on the beneficial effects of fibrin gel network (L-PRF) made of various cells leukocytes, erythrocytes and platelets for improving the SC proliferation and neurotrophic secretion[42]. Although this study has not investigated particularly HPL and its effects on ASC and axonal growth from neuronal cells, observations made at cellular and molecular level would further support and strengthen the results obtained and some of the underlying mechanisms hypothesized in our study. Furthermore, the robust axonal outgrowth observed in the continuous supplementation of HPL, in other words, not only ASC’s preconditioning, but also to the DRG and ASC co-cultures may be explained by the paracrine signaling established through the cross talk between factors derived from regenerating axons and ASC.

These mechanisms need to be elucidated along with the secretome profile of the HPL-conditioned ASC in order to gain more insights into the intra-cellular mechanisms. In a continuous note, the molecular structure of the cytoskeleton and electrophysiological function of regenerated axons, supported by HPL-stimulated ASC, remain to be analyzed.

Conclusion

Our study reports for the first time the enhanced neurotrophic potency of human adipose stem cells in response to HPL-conditioning for promoting the axonal outgrowth to the level comparable to the gold standard SC. In a short note, the positive effects found on axonal regeneration are result of a synergic effect of the HPL-culture medium and the HPL-preconditioned ASC. These findings provide new knowledge and insights for developing the adipose stem cell-based therapies in the field of nerve tissue engineering. Furthermore, HPL-based strategies may potentially be implemented from autologous source, which may open the options for personalized treatment and clinical translation.

Future perspective

Culture, expansion andex vivostimulation of stem cells using the additive therapeutics of autologous origin holds a great promise for personalized medicine. In the present study, we demonstrated significantly enhanced neurotrophic ability for HPL-stimulated human ASC for promoting the axonal outgrowth. Thus, our approach involving the

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second, a viable cell source matching with SC in the context of axonal regeneration. SC are the gold standard for supporting nerve regeneration, although their clinical usability is limited due to inevitable problems associated with their access, culture and expansion. Taken together, HPL-stimulated adipose stem cells would represent a potential option for implementation of autologous therapy and clinical translation.

Author contributions

SM contributed to the conception and design of the study; M Lischer and PG di Summa conducted the experiments; S Madduri, DF Kalbermatten, R Guzman, DJ Schaefer, CM Oranges and M Lischer organized the data collection and analysis; S Madduri and M Lischer performed the statistical analysis; S Madduri and M Lischer wrote the manuscript and S Madduri revised the manuscript. All the authors contributed to manuscript revision and approved the manuscript submission.

Acknowledgments

The authors thank M Abanto and L Sauteur of the DBM Microscopy core Facility, University of Basel for the excellent technical support.

Financial & competing interests disclosure

The authors thank the enabling research grant support by EUROSTAR to S Madduri and by surgery foundation of University Hospital Basel to S Madduri, DF Kalbermatten and R Guzman. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

All materials with human origin were obtained in accordance with the ethical standards of the Institutional Ethics Committee and Institutional Committee for Stem Cell Research of University Hospital Basel. Informed consent was obtained from the involved participants.

Summary points

Human platelet lysate (HPL) supplemented growth medium promoted the important axonal outgrowth from the dorsal root ganglion explants, in contrast to fetal bovine serum.

HPL-stimulated adipose stem cells exhibited significantly enhanced neurotrophic potency as evidenced by robust axonal outgrowth.

HPL-stimulated adipose stem cells appeared to match with Schwann cells in the context of axonal regenerationin vitro.

Regime analysis of supplementation of growth medium additives revealed key role of HPL in dorsal root ganglions and stem cell co-cultures for mounting effective axonal regeneration.

References

1. Evans GR. Peripheral nerve injury: a review and approach to tissue engineered constructs.Anat. Rec.263(4), 396–404 (2001).

2. Bruyns CN, Jaquet JB, Schreuders TA, Kalmijn S, Kuypers PD, Hovius SE. Predictors for return to work in patients with median and ulnar nerve injuries.J. Hand Surg. (Am.)28(1), 28–34 (2003).

3. Mohanna PN, Young RC, Wiberg M, Terenghi G. A composite poly-hydroxybutyrate-glial growth factor conduit for long nerve gap repairs.J. Anat.203(6), 553–565 (2003).

4. Dvali L, Mackinnon S. Nerve repair, grafting, and nerve transfers.Clin. Plast. Surg.30(2), 203–221 (2003).

5. Wiberg M, Terenghi G. Will it be possible to produce peripheral nerves?Surg. Technol. Int.11, 303–310 (2003).

6. Bain JR, Mackinnon SE, Hudson ARet al.The peripheral nerve allograft in the primate immunosuppressed with cyclosporin A: i.

histologic and electrophysiologic assessment.Plast. Reconstr. Surg.90(6), 1036–1046 (1992).

7. Heath CA, Rutkowski GE. The development of bioartificial nerve grafts for peripheral-nerve regeneration.Trends Biotechnol.16(4), 163–168 (1998).

8. Hu J, Zhu QT, Liu XL, Xu YB, Zhu JK. Repair of extended peripheral nerve lesions in rhesus monkeys using acellular allogenic nerve grafts implanted with autologous mesenchymal stem cells.Exp. Neurol.204(2), 658–666 (2007).

(11)

9. Jessen KR, Mirsky R. Schwann cells and their precursors emerge as major regulators of nerve development.Trends Neurosci.22(9), 402–410 (1999).

10. Di Summa PG, Kalbermatten DF, Pralong E, Raffoul W, Kingham PJ, Terenghi G. Long-termin vivoregeneration of peripheral nerves through bioengineered nerve grafts.Neuroscience181, 278–291 (2011).

11. Mosahebi A, Fuller P, Wiberg M, Terenghi G. Effect of allogeneic Schwann cell transplantation on peripheral nerve regeneration.Exp.

Neurol.173(2), 213–223 (2002).

12. Xue C, Hu N, Gu Yet al.Joint use of a chitosan/PLGA scaffold and MSCs to bridge an extra large gap in dog sciatic nerve.

Neurorehabil. Neural Repair26(1), 96–106 (2012).

13. Casanas J, De La Torre J, Soler Fet al.Peripheral nerve regeneration after experimental section in ovine radial and tibial nerves using synthetic nerve grafts, including expanded bone marrow mesenchymal cells: morphological and neurophysiological results.Injury 45(Suppl. 4), S2–S6 (2014).

14. Debnath T, Chelluri LK. Standardization and quality assessment for clinical grade mesenchymal stem cells from human adipose tissue.

Hematol. Transfus. Cell Ther.41(1), 7–16 (2019).

15. Strem BM, Hicok KC, Zhu Met al.Multipotential differentiation of adipose tissue-derived stem cells.Keio J. Med.54(3), 132–141 (2005).

16. Kern S, Eichler H, Stoeve J, Kluter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue.Stem Cells24(5), 1294–1301 (2006).

17. Nakagami H, Maeda K, Morishita Ret al.Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells.Arterioscler. Thromb. Vasc. Biol.25(12), 2542–2547 (2005).

18. El Atat O, Antonios D, Hilal Get al.An evaluation of the stemness, paracrine, and tumorigenic characteristics of highly expanded, minimally passaged adipose-derived stem cells.PLoS ONE11(9), e0162332 (2016).

19. Wang X, Luo E, Li Y, Hu J. Schwann-like mesenchymal stem cells within vein graft facilitate facial nerve regeneration and remyelination.

Brain Res.1383, 71–80 (2011).

20. Watanabe Y, Sasaki R, Matsumine H, Yamato M, Okano T. Undifferentiated and differentiated adipose-derived stem cells improve nerve regeneration in a rat model of facial nerve defect.J. Tissue Eng. Regen. Med.11(2), 362–374 (2017).

21. Doucet C, Ernou I, Zhang Yet al.Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.J. Cell. Physiol.205(2), 228–236 (2005).

22. Bernardi M, Agostini F, Chieregato Ket al.The production method affects the efficacy of platelet derivatives to expand mesenchymal stromal cellsin vitro.J. Transl. Med.15(1), 90 (2017).

23. Dessels C, Potgieter M, Pepper MS. Making the switch: alternatives to fetal bovine serum for adipose-derived stromal cell expansion.

Front. Cell Dev. Biol.4, 115 (2016).

24. Kakudo N, Morimoto N, Ma Y, Kusumoto K. Differences between the proliferative effects of human platelet lysate and fetal bovine serum on human adipose-derived stem cells.Cells8(10), 1218 (2019).

25. Horn P, Bokermann G, Cholewa Det al.Impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells.Cytotherapy12(7), 888–898 (2010).

26. Bieback K, Hecker A, Koca¨omer Aet al.Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow.Stem Cells27(9), 2331–2341 (2009).

27. Hemeda H, Giebel B, Wagner W. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

Cytotherapy16(2), 170–180 (2014).

28. Kingham PJ, Kalbermatten DF, Mahay D, Armstrong SJ, Wiberg M, Terenghi G. Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowthin vitro.Exp. Neurol.207(2), 267–274 (2007).

29. Pareek S, Suter U, Snipes GJ, Welcher AA, Shooter EM, Murphy RA. Detection and processing of peripheral myelin protein PMP22 in cultured Schwann cells.J. Biol. Chem.268(14), 10372–10379 (1993).

30. Atanasoski S, Notterpek L, Lee HYet al.The protooncogene Ski controls Schwann cell proliferation and myelination.Neuron43(4), 499–511 (2004).

31. Madduri S, Papaloizos M, Gander B. Synergistic effect of GDNF and NGF on axonal branching and elongationin vitro.Neurosci. Res.

65(1), 88–97 (2009).

32. Georgiou M, Golding JP, Loughlin AJ, Kingham PJ, Phillips JB. Engineered neural tissue with aligned, differentiated adipose-derived stem cells promotes peripheral nerve regeneration across a critical sized defect in rat sciatic nerve.Biomaterials37, 242–251 (2015).

33. Bucan V, Vaslaitis D, Peck CT, Strauss S, Vogt PM, Radtke C. Effect of exosomes from rat adipose-derived mesenchymal stem cells on neurite outgrowth and sciatic nerve regeneration after crush injury.Mol. Neurobiol.56(3), 1812–1824 (2019).

34. Klein SM, Vykoukal J, Li DPet al.Peripheral motor and sensory nerve conduction following transplantation of undifferentiated autologous adipose tissue-derived stem cells in a biodegradable U.S. Food and Drug Administration-approved nerve conduit.Plast.

Reconstr. Surg.138(1), 132–139 (2016).

(12)

36. Orbay H, Uysal AC, Hyakusoku H, Mizuno H. Differentiated and undifferentiated adipose-derived stem cells improve function in rats with peripheral nerve gaps.J. Plast. Reconstr. Aesthet. Surg.65(5), 657–664 (2012).

37. Hemeda H, Kalz J, Walenda G, Lohmann M, Wagner W. Heparin concentration is critical for cell culture with human platelet lysate.

Cytotherapy15(9), 1174–1181 (2013).

38. Blair P, Flaumenhaft R. Platelet alpha-granules: basic biology and clinical correlates.Blood Rev.23(4), 177–189 (2009).

39. Schallmoser K, Bartmann C, Rohde Eet al.Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.Transfusion47(8), 1436–1446 (2007).

40. Huang CT, Chu HS, Hung KCet al.The effect of human platelet lysate on corneal nerve regeneration.Br. J.

Ophthalmol.doi:10.1136/bjophthalmol-2019-314408 (2019) (Epub ahead of print).

41. Dezawa M, Takahashi I, Esaki M, Takano M, Sawada H. Sciatic nerve regeneration in rats induced by transplantation ofin vitro differentiated bone-marrow stromal cells.Eur. J. Neurosci.14(11), 1771–1776 (2001).

42. Wang Z, Mudalal M, Sun Yet al.The effects of leukocyte-platelet rich fibrin (L-PRF) on suppression of the expressions of the pro-inflammatory cytokines, and proliferation of Schwann cell, and neurotrophic factors.Sci. Rep.10(1), 2421 (2020).

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