Human platelet lysate as a potential clinical-translatable supplement to support the neurotrophic properties of human adipose-derived stem cells

Abstract

Background: The autologous nerve graft, despite its donor site morbidity and unpredictable functional recovery, continues to be the gold standard in peripheral nerve repair. Rodent research studies have shown promising results with cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit, as an alternative strategy for nerve regeneration. To achieve meaningful clinical translation, cell therapy must comply with biosafety. Cell extraction and expansion methods that use animal-derived products, including enzymatic adipose tissue dissociation and the use of fetal bovine serum (FBS) as a culture medium supplement, have the potential for transmission of zoonotic infectious and immunogenicity. Human-platelet-lysate (hPL) serum has been used in recent years in human cell expansion, showing reliability in clinical applications.

Methods: We investigated whether hADSC can be routinely isolated and cultured in a completely xenogeneic-free way (using hPL culture medium supplement and avoiding collagenase digestion) without altering their physiology and stem properties. Outcomes in terms of stem marker expression (CD105, CD90, CD73) and the osteocyte/adipocyte differentiation capacity were compared with classical collagenase digestion and FBS-supplemented hADSC expansion.

Results: We found no significant differences between the two examined extraction and culture protocols in terms of cluster differentiation (CD) marker expression and stem cell plasticity, while hADSC in hPL showed a significantly higher proliferation rate when compared with the usual FBS-added medium. Considering the important key growth factors (particularly brain-derived growth factor (BDNF)) present in hPL, we investigated a possible neurogenic commitment of hADSC when cultured with hPL. Interestingly, hADSC cultured in hPL showed a statistically higher secretion of neurotrophic factors BDNF, glial cell-derived growth factor (GDNF), and nerve-derived growth factor (NFG) than FBS-cultured cells. When cocultured in the presence of primary neurons, hADSC which had been grown under hPL supplementation, showed significantly enhanced neurotrophic properties.

Conclusions: The hPL-supplement medium could improve cell proliferation and neurotropism while maintaining stable cell properties, showing effectiveness in clinical translation and significant potential in peripheral nerve research.

Keywords: Cell therapy; Human adipose-derived stem cells (hADSC); Human platelet lysate (hPL); Peripheral nerve injury (PNI).

Fig. 1

Morphology and proliferation of hADSC in the four different conditions. a Representative phase-contrast pictures of hADSC cultured with FBS (left column) or hPL (right column) after isolation with collagenase (upper row) or with explant method (lower row). b Cell proliferation was evaluated measuring metabolic activity of hADSC from days 3 to 7 after seeding. Experiments were conducted in three technical and biological (3 donors) repeats. One-way ANOVA multiple comparisons tests were used for proliferation assays to assess statistical significance among the four examined extraction/culture conditions: (p < 0.05)

Fig. 2

Differentiation of hADSC in the four different conditions. a Pictures representing the good adipogenic differentiation rate of hADSC cultured in all conditions. b Osteogenic differentiation of hADSC occurred only with hPL-based cultures (right panels) but not with FBS cultures (left panels). Insets show control cells without differentiation medium. Black scale bars indicate 100 μm. Experiments were conducted in three technical and three biological (3 donors) repeats for adipogenic differentiation while in 5 biological replicates for the osteogenic one

Fig. 3

Evaluation of SC markers in hADSC in the two conditions. a Evaluation of secreted factors (NGF, BDNF, and GDNF) by hADSC cultured in classical and cell therapy-ready conditions. One-way ANOVA multiple comparisons tests were used for assessing statistical significance among the examined conditions: (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). b Gene expression of hADSC surface receptors, NTRK1 (coding for the high-affinity receptor for NGF), NTRK2 (coding for the high-affinity receptor for BDNF), NGFR (coding for the low-affinity receptor for NGF and BDNF), and GFRA1 (coding for the receptor for GDNF). c Representative fluorescence pictures of intracellular spotted signal detection of STRO-1, Nestin, GFAP, and MPZ in hADSC cultured in classical (left panel) and cell therapy-ready conditions (right panel). Experiments were conducted in three technical and biological (3 donors) repeats

Fig. 4

Functional analysis using an in vitro co-culture model with DRG. Immunofluorescence microscopy (anti-ß3-Tubulin) of DRG alone (a FBS, d hPL), indirect co-culture (b FBS cond med, e hPL cond med), and direct co-culture serum-free (c ADSC (FBS-free), f ADSC (hPL-free)). Neurite maximal length (g) and neurite area (h) of single DRG grown alone, in direct or indirect co-culture with hADSC expanded in FBS or hPL. i Neurite maximal length of direct co-culture of single DRG with hADSC expanded in FBS or hPL—comparison between no serum-free and serum-free co-cultures. Scale bar: 500 μm. Experiments were conducted in biological triplicates (3 hADSC donors) and 9 technical repeats one-way ANOVA with Tukey’s multiple comparison tests were used for assessing statistical significance among the examined conditions: (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)