In vitro Characteristics of Heterogeneous Equine Hoof Progenitor Cell Isolates

Abstract

Damage to an ectodermal-mesodermal interface like that in the equine hoof and human finger nail bed can permanently alter tissue structure and associated function. The purpose of this study was to establish and validate in vitro culture of primary progenitor cell isolates from the ectodermal-mesodermal tissue junction in equine hooves, the stratum internum, with and without chronic inflammation known to contribute to lifelong tissue defects. The following were evaluated in hoof stratum internum cell isolates up to 5 cell passages (P): expansion capacity by cell doublings and doubling time; plasticity with multi-lineage differentiation and colony-forming unit (CFU) frequency percentage; immunophenotype with immunocytochemistry and flow cytometry; gene expression with RT-PCR; and ultrastructure with transmission electron microscopy. The presence of keratin (K)14, 15 and K19 as well as cluster of differentiation (CD)44 and CD29 was determined in situ with immunohistochemistry. To confirm in vivo extracellular matrix (ECM) formation, cell-scaffold (polyethylene glycol/poly-L-lactic acid and tricalcium phosphate/hydroxyapatite) constructs were evaluated with scanning electron microscopy 9 weeks after implantation in athymic mice. Cultured cells had characteristic progenitor cell morphology, expansion, CFU frequency percentage and adipocytic, osteoblastic, and neurocytic differentiation capacity. CD44, CD29, K14, K15 and K19 proteins were present in native hoof stratum internum. Cultured cells also expressed K15, K19 and desmogleins 1 and 3. Gene expression of CD105, CD44, K14, K15, sex determining region Y-box 2 (SOX2) and octamer-binding transcription factor 4 (OCT4) was confirmed in vitro . Cultured cells had large, eccentric nuclei, elongated mitochondria, and intracellular vacuoles. Scaffold implants with cells contained fibrous ECM 9 weeks after implantation compared to little or none on acellular scaffolds. In vitro expansion and plasticity and in vivo ECM deposition of heterogeneous, immature cell isolates from the ectodermal-mesodermal tissue interface of normal and chronically inflamed hooves are typical of primary cell isolates from other adult tissues, and they appear to have both mesodermal and ectodermal qualities in vitro . These results establish a unique cell culture model to target preventative and restorative therapies for ectodermal-mesodermal tissue junctions.

Equine Hoof Stem Progenitor Cells (HPC) CD29 + /Nestin + /K15 + – a Novel Dermal/epidermal Stem Cell Population With a Potential Critical Role for Laminitis Treatment

“…The animals were euthanized for reasons unrelated to this study. The tissue was dissected in sterile conditions with a scalpel blade and placed in Dulbecco's modified Eagle's medium/ F12 (DMEM/F12, Sigma Aldrich/Merck, Poznan, Poland) supplemented with 1% penicillin/streptomycin mix (P/S, Sigma Aldrich, Munich, Germany).Within 1,5 h of harvest, EHSPC cells were isolated and cultured, following the protocol previously described by Yang et al [14]. The osteogenic and chondrogenic differentiation were induced using StemPro™ Osteogenesis Differentiation Kit and StemPro™ Choondrogenesis Differentiation Kit(Thermo Fisher Scientific, Warsaw, Poland).…”

The Equine Hoof: Laminitis, Progenitor (Stem) Cells, and Therapy Development

“…Current knowledge supports that primary progenitor cell isolates from normal and abnormal equine hoof stratum lamellatum express ectodermal (K14, K15, K19) and mesodermal (CD29, CD44, CD105) genes and proteins, and they have both ectodermal (neurogenic) and mesodermal (osteogenic, adipogenic) differentiation abilities. 135 Further, equine hoof progenitor cells share some ultrastructural features of both epidermal and mesodermal equine progenitor cells like intracellular vacuoles and well-developed dilated rough endoplasmic reticulum cisternae (mesodermal), as well as cell surface microvilli (ectodermal). 136,137 The cells also produce extracellular matrix on scaffold carriers composed of either polyester or mineral and on decellularized lamellar matrix in vivo, confirming the ability to form neotissue on custom templates.…”

“…136,137 The cells also produce extracellular matrix on scaffold carriers composed of either polyester or mineral and on decellularized lamellar matrix in vivo, confirming the ability to form neotissue on custom templates. 115,135 This information suggests that in vitro cell models of the equine stratum internum may be an alternative to in vivo induction of laminitis in otherwise normal hooves.…”

“…155 Recently, cells that express epidermal (K15) or mesodermal (CD105) proteins were identified in the stratum lamellatum. 156 Progenitor cell isolates that expressed both proteins were subsequently isolated from heterogenous stratum lamellatum primary cell isolates and cultured through multiple passages in vitro (Figure 10). 135 The cells showed ectodermal and mesodermal plasticity and transdifferentiation capabilities.…”

“…156 Progenitor cell isolates that expressed both proteins were subsequently isolated from heterogenous stratum lamellatum primary cell isolates and cultured through multiple passages in vitro (Figure 10). 135 The cells showed ectodermal and mesodermal plasticity and transdifferentiation capabilities. The potential for EMT to contribute to laminitis is suggested by the abundant mesodermal wedge of fibrous tissue typical of the condition.…”