Ultrastructural morphology is distinct among primary progenitor cell isolates from normal, inflamed, and cryopreserved equine hoof tissue and CD105(+)K14(+) progenitor cells

The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning electron microscopy, immunocytochemistry, and CD105(+)K14(+) cell trilineage plasticity. Cells from normal tissue had typical progenitor cell characteristics. Those from inflamed tissue had organelles and morphology consistent with catabolic activities including lysosomes, irregular rough endoplasmic reticulum, and fewer vacuoles and early endosomes than those from normal tissue. Cryopreserved tissue cells appeared apoptotic with an irregular cell membrane covered by cytoplasmic protrusions closely associated with endocytic and exocytic vesicles, chromatin aggregated on the nuclear envelop, abundant, poorly organized rough endoplasmic reticulum, and plentiful lysosomes. Cells that were CD105(+)K14(+) were distinguishable from heterogenous cells by infrequent microvilli on the cell surface, sparse endosomes and vesicles, and desmosomes between cells. Cells expressed ectodermal (K15) and mesodermal (CD105) proteins in 2D and 3D cultures. Inflamed and cryopreserved tissue isolates attached poorly to tissue scaffold while normal tissue cells attached well, but only CD105(+)K14(+) cells produced extracellular matrix after 4 d. The CD105(+)K14(+) cells exhibited osteoblastic, adipocytic, and neurocytic differentiation. Ultrastructural information provided by this study contributes to understanding of equine hoof progenitor cells to predict their potential contributions to tissue maintenance, healing, and damage as well post-implantation behavior.