Differentiation of equine induced pluripotent stem cells into a keratinocyte lineage.

REASONS FOR PERFORMING STUDY: Skin trauma in horses often leads to the development of chronic nonhealing wounds that lack a keratinocyte cover, vital for healing. Reports in mouse and man confirm the possibility of generating functional keratinocytes from induced pluripotent stem cells (iPSC), thus presenting myriad potential applications for wound management or treatment of skin disease. Similarly, differentiation of equine iPSC (eiPSC) into a keratinocyte lineage should provide opportunities for the advancement of veterinary regenerative medicine. OBJECTIVES: The purpose of this study was to develop an efficient method for the differentiation of eiPSC into a keratinocyte lineage. It was hypothesised that eiPSC can form differentiated keratinocytes (eiPSC-KC) comparable with primary equine keratinocytes (PEK) in their morphological and functional characteristics. STUDY DESIGN: Experimental in vitro study. METHODS: Equine iPSC established using a nonviral system were treated for 30 days with retinoic acid and bone morphogenetic protein-4 to induce directed differentiation into iPSC-KC. Temporospatial gene and protein expression by eiPSC-KC was measured at weekly intervals of differentiation and in response to calcium switch. Proliferative and migratory capacities of eiPSC-KC were compared with those of PEK. RESULTS: Equine iPSC, upon directed differentiation, showed loss of pluripotency genes and progressive increase in pancytokeratin expression indicating ectodermal specification into keratinocytes. High differentiation efficiency was achieved, with 82.5% of eiPSC expressing keratin 14, a marker of epidermal-specific basal stem cells, after 30 days of directed differentiation. Moreover, the proliferative capacity of eiPSC-KC was superior, while the migratory capacity (measured as the ability to epithelise in vitro wounds) was comparable with that of PEK. CONCLUSIONS: This proof of concept study suggests that eiPSC can successfully be differentiated into equine keratinocytes (eiPSC-KC) with features that are promising to the development of a stem cell-based skin construct, with the potential to regenerate lost or damaged skin.

Immune potential of allogeneic equine induced pluripotent stem cells.

REASONS FOR PERFORMING STUDY: Induced pluripotent stem cells (iPSC) have brought immense hope to cellular therapy and regenerative medicine. However, the antigenicity of iPSC has not been well documented and remains a hurdle for clinical applications. Expression of major histocompatibility complex (MHC) molecules by human and murine iPSC is downregulated, making these cells potentially safe for transplantation. No such data are available for any large animal model. OBJECTIVES: To measure expression of MHC molecules on equine iPSC (eiPSC) and describe their antigenicity using intradermal testing. The hypothesis was that allogeneic eiPSC weakly express MHC molecules and would not elicit a rejection response when injected intradermally. STUDY DESIGN: Experimental study involving both in vitro and in vivo components. METHODS: Two green fluorescent protein-expressing eiPSC lines were analysed by flow cytometry for MHC expression. One line was then transplanted intradermally, along with appropriate controls, into 2 unrelated experimental horses. Blood was collected pre- and 7 days post transplantation. The wheals formed at the sites of injection were measured at regular intervals beginning at 0.25 h until 4 weeks. Tissue samples of the injected sites were obtained at 2, 3, 7 and 30 days post transplantation and analysed by histopathology and immunofluorescence. RESULTS: Both eiPSC lines weakly expressed MHC molecules. eiPSC were detectable up to 7 days following allogeneic transplantation and elicited no apparent systemic response. Injection of eiPSC caused small wheal formation at the skin surface. Skin sections revealed CD4(+) and CD8(+) mononuclear cells up to 30 days post transplantation. CONCLUSIONS: These data suggest that while transplantation of allogeneic eiPSC elicits a moderate cellular response, it does not cause acute rejection. The feasibility of banking allogeneic iPSC for regenerative medicine applications should be explored.

The application of quantitative oil streaking to the HeartQuest left ventricular assist device.

Methods of flow visualization using oil streaking are established techniques for investigating surface shear and near wall flow patterns. Recent studies have used an array of oil dots on a surface which form streaks when exposed to shear forces. This method is generally qualitative, but it is possible to make quantitative measurements of the shear if the oil streaks have been calibrated. This paper presents the application of a quantitative oil streak method to the HeartQuest left ventricular assist device (LVAD). An array of dots was applied to the top housing of the pump, yielding quantitative values for the shear and qualitative patterns of the near wall flow in that region. The results were used to locate regions likely to promote thrombosis, such as stagnation points or recirculation regions. Regions of high shear, where hemolysis might occur, also can be identified with this method. In addition to being an important design technique, quantitative oil streaking assisted in the verification of computational fluid dynamics results within the HeartQuest LVAD.

Differentiation of radiation-induced fibrosis from recurrent pulmonary neoplasm by CT.

We describe and compare the computed tomographic (CT) manifestations of radiation fibrosis and recurrent neoplasm and consider the potential application of CT in the follow-up of patients after radiotherapy for bronchogenic carcinoma. Thirty-one patients were evaluated in this retrospective study. The CT findings in 20 patients with radiation fibrosis were compared with those in 11 patients with recurrent tumor. In 19 of the 20 patients with radiation fibrosis, CT allowed demonstration of consolidation, with a straight lateral margin and containing ectatic air-containing bronchi. In 9 of the 11 patients with recurrent tumor, CT showed a soft tissue mass with a convex lateral border and without air-containing bronchi. Twenty of the 31 patients had a suspected recurrence on plain chest radiographs. CT permitted accurate differentiation between radiation fibrosis and recurrent tumor in all patients, with the exception of two who had large pleural effusions. Because of its great specificity, CT appears useful in complementing chest radiographs in the follow-up of patients after radiotherapy.

Computed tomography used to exclude pneumothorax in bullous lung disease.

We report two patients with bullous lung disease and suspected pneumothorax on plain chest radiographs. Chest tubes were inserted in both patients, but no clinical or radiological improvement occurred. Computed tomographic (CT) scans were then performed and showed bullous lesions without any pneumothorax. This permitted the removal of unnecessary chest tubes. In selected patients with severe bullous disease, CT may be useful, either on admission or later, when the diagnosis of pneumothorax by conventional radiographs is doubtful.

Traumatic subarachnoid-mediastinal fistula mimicking a ruptured aorta.

A 35-year-old man, injured in an automobile accident was found to have a subarachnoid-mediastinal fistula that mimicked a ruptured aorta. Of 16 reported cases of traumatic subarachnoid-mediastinal fistula, this is only the third in which the fistulous communication was with the extrapleural space only. The possibility of subarachnoid-mediastinal fistula, although rare, should be entertained in injuries to the thoracocervical spine when neurologic symptoms are present.