Human umbilical cord mesenchymal stromal cells exhibit immature nucleus pulposus cell phenotype in a laminin-rich pseudo-three-dimensional culture system

Abstract

IntroductionCell supplementation to the herniated or degenerated intervertebral disc (IVD) is a potential strategy to promote tissue regeneration and slow disc pathology. Human umbilical cord mesenchymal stromal cells (HUCMSCs) – originating from the Wharton’s jelly – remain an attractive candidate for such endeavors with their ability to differentiate into multiple lineages. Previously, mesenchymal stem cells (MSCs) have been studied as a potential source for disc tissue regeneration. However, no studies have demonstrated that MSCs can regenerate matrix with unique characteristics matching that of immature nucleus pulposus (NP) tissues of the IVD. In our prior work, immature NP cells were found to express specific laminin isoforms and laminin-binding receptors that may serve as phenotypic markers for evaluating MSC differentiation to NP-like cells. The goal of this study is to evaluate these markers and matrix synthesis for HUCMSCs cultured in a laminin-rich pseudo-three-dimensional culture system.MethodsHUCMSCs were seeded on top of Transwell inserts pre-coated with Matrigel™, which contained mainly laminin-111. Cells were cultured under hypoxia environment with three differentiation conditions: NP differentiation media (containing 2.5% Matrigel™ solution to provide for a pseudo-three-dimensional laminin culture system) with no serum, or the same media supplemented with either insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1). Cell clustering behavior, matrix production and the expression of NP-specific laminin and laminin-receptors were evaluated at days 1, 7, 13 and 21 of culture.ResultsData show that a pseudo-three-dimensional culture condition (laminin-1 rich) promoted HUCMSC differentiation under no serum conditions. Starting at day 1, HUCMSCs demonstrated a cell clustering morphology similar to that of immature NP cells in situ and that observed for primary immature NP cells within the similar laminin-rich culture system (prior study). Differentiated HUCMSCs under all conditions were found to contain glycosaminoglycan, expressed extracellular matrix proteins of collagen II and laminin α5, and laminin receptors (integrin α3 and β4 subunits). However, neither growth factor treatment generated distinct differences in NP-like phenotype for HUCMSC as compared with no-serum conditions.ConclusionsHUCMSCs have the potential to differentiate into cells sharing features with immature NP cells in a laminin-rich culture environment and may be useful for IVD cellular therapy.

Inflammation in intervertebral disc degeneration and regeneration

“…Several cell-based therapies to stimulate IVD regeneration have been proposed in recent years: haematopoietic stem cells (HSC) [186], fetal spine cells [187], immortalized NP-cell lines [188], autologous IVD chondrocytes [189], embryonic stem cells (SC) [190], induced pluripotent SC [191], olfactory SC [192] and MSCs (derived either from bone marrow [193] or from umbilical cord blood [194]) have all been suggested to have potential for IVD repair/regeneration. NP progenitor cells were isolated from the NP (with approximately 1% frequency) and differentiated into chondrogenic and neurogenic lineages, suggesting potential for IVD regeneration [195].…”

Advancing biomaterials of human origin for tissue engineering

“…Similarly, when a PEG-based hydrogel was functionalized with the laminin-derived adhesion peptide YIGSR, the resulting biomaterial significantly enhanced intracellular triglyceride accumulation in encapsulated adipocytes and ultimately promoted the formation of coherent adipose tissue-like structures featuring many mature unilocular fat cells [361]. Although MSCs have been suggested to be a potential source for disc tissue regeneration, these cells neither differentiate into NP-like cells nor regenerate matrix with unique characteristics matching that of immature NP tissues of the IVD unless co-cultured with human NP cells or placed in a laminin-rich culture environment [362,363]. Indeed, substantial evidence suggests that NP cell-laminin interplay is unique to the immature disc because immature NP cells were found to express specific laminin isoforms and laminin-binding receptors [364,365].…”

Cell therapy for the degenerating intervertebral disc

“…Mesenchymal stem cells (MSCs) from various sources (e.g., bone marrow, fat,(28) umbilical cord blood,(22) Wharton's jelly,(23, 75) olfactory stem cells(42)) or induced pluripotent stem cells (76) have also been investigated for repairing the degenerate IVD. While readily available, MSCs may suffer from overt cell loss when implanted in the undifferentiated state, due to inability to survive in the harsh, nutrient-poor environment.…”