Xeno-Free Spheroids of Human Gingiva-Derived Progenitor Cells for Bone Tissue Engineering

Abstract

Gingiva has been identified as a minimally invasive source of multipotent progenitor cells (GPCs) for use in bone tissue engineering (BTE). To facilitate clinical translation, it is important to characterize GPCs in xeno-free cultures. Recent evidence indicates several advantages of three-dimensional (3D) spheroid cultures of mesenchymal stromal cells (MSCs) over conventional 2D monolayers. The present study aimed to characterize human GPCs in xeno-free 2D cultures, and to test their osteogenic potential in 3D cultures, in comparison to bone marrow MSCs (BMSCs). Primary GPCs and BMSCs were expanded in human platelet lysate (HPL) or fetal bovine serum (FBS) and characterized based on in vitro proliferation, immunophenotype and multilineage differentiation. Next, 3D spheroids of GPCs and BMSCs were formed via self-assembly and cultured in HPL. Expression of stemness-(SOX2, OCT4, NANOG) and osteogenesis-related markers (BMP2, RUNX2, OPN, OCN) was assessed at gene and protein levels in 3D and 2D cultures. The cytokine profile of 3D and 2D GPCs and BMSCs was assessed via a multiplex immunoassay. Monolayer GPCs in both HPL and FBS demonstrated a characteristic MSC-like immunophenotype and multi-lineage differentiation; osteogenic differentiation of GPCs was enhanced in HPL vs. FBS. CD271 + GPCs in HPL spontaneously acquired a neuronal phenotype and strongly expressed neuronal/glial markers. 3D spheroids of GPCs and BMSCs with high cell viability were formed in HPL media. Expression of stemness-and osteogenesisrelated genes was significantly upregulated in 3D vs. 2D GPCs/BMSCs; the latter was independent of osteogenic induction. Synthesis of SOX2, BMP2 and OCN was confirmed via immunostaining, and in vitro mineralization via Alizarin red staining. Finally, secretion of several growth factors and chemokines was enhanced in GPC/BMSC spheroids, while that of pro-inflammatory cytokines was reduced, compared to monolayers. In summary, monolayer GPCs expanded in HPL demonstrate enhanced osteogenic differentiation potential, comparable to that of BMSCs. Xeno-free spheroid culture further enhances stemness-and osteogenesis-related gene expression, and cytokine secretion in GPCs, comparable to that of BMSCs.

Spheroid Coculture of Human Gingiva-Derived Progenitor Cells With Endothelial Cells in Modified Platelet Lysate Hydrogels

“…In contrast to 2D monolayers, 3D spheroid culture has been reported to enhance survival, stemness, paracrine activity, immunomodulation and multi-lineage differentiation of MSC (Follin et al, 2016;Petrenko et al, 2017) (Kale et al, 2000;Chatterjea et al, 2017). In the context of BTE applications, we have observed particular advantages of spheroid vs monolayer culture via a strong upregulation of osteogenesis-related genes in BMSC and GPC (Shanbhag et al, 2020b).…”

“…Monolayer cultures of primary human GPC isolated from healthy donors were established in 5% HPL (Bergenlys ® , Bergen, Norway). Details of isolation and characterization of GPC have been reported elsewhere (Shanbhag et al, 2020b). Early passage human umbilical vein EC (HUVEC) were purchased and cultured in EGM-2 growth medium (both from Lonza Inc., Walkersville, United States) supplemented with either 2% FBS, as per the manufacturer's recommendations, or with 5% HPL; all other media components were maintained.…”

“…3D aggregate spheroids of HUVEC were formed via guided selfassembly in microwell plates as recently described (Shanbhag et al, 2020b). Briefly, suspensions of dissociated monolayer HUVEC cultured in FBS or HPL, were seeded in microwell plates (Sphericalplate ® , Kugelmeiers Ltd., Erlenbach, Switzerland) for 24 h to form spheroids of ∼1000 cells each.…”

“…Accordingly, xeno-free alternatives such as pooled human platelet lysate (HPL), have emerged and have been shown to be comparable, and often superior, to FBS for the proliferation and differentiation of various types of MSC (Fekete et al, 2012;Shanbhag et al, 2017b;Shanbhag et al, 2020a). We have recently reported that xeno-free culture of human GPC in HPL vs FBS media results in enhanced growth, gene expression and differentiation (Shanbhag et al, 2020b). Moreover, the proliferation and tube formation of EC is reported to be enhanced in HPL (Tasev et al, 2015) and other xeno-free media (Bauman et al, 2018).…”

Gingiva-Derived Mesenchymal Stem Cells: Potential Application in Tissue Engineering and Regenerative Medicine - A Comprehensive Review

“…More importantly, systemic administration of 3D-spheroid GMSCs display enhanced therapeutic effects on chemotherapy-induced oral mucositis in mice as compared to their 2D-adherent counterparts (115). Similarly, 3D-spheroid GMSCs generated under different culture conditions also exhibit enhanced stemness, trilineage and neural differentiation potentials, and cytokine secretion (152,156). Several studies show that GMSCs cultured in patterned microwells can aggregate into 3D-spheoids with enhanced osteogenic potentials, which are further augmented by the addition of lovastatin or FGF-4 (153)(154)(155).…”