Exploring epidermal stem cell engraftment

Objective: Cultured autologous epidermal stem cells are used to treat extensively burned patients. However, engraftment is variable and it is fundamental to know 1- how many stem cells survive the stress of transplantation and 2- how many stem cells are needed for long-term self-renewal of the regenerated epidermis. Therefore, we have recapitulated the transplantation of autologous cultured epidermal stem cells in the minipig to investigate the cellular and molecular mechanisms involved in engraftment. Methods: Pig keratinocytes were cultivated according to the protocol used in human epidermal cell therapy. Human surgical procedures were adapted to the pig. Engraftment was evaluated clinically and by histology. The presence of epidermal stem cells was evaluated by clonal analysis. The presence of dividing or apoptotic cells was revealed by Ki67 and cleaved-caspase3 immunostaining respectively. Results: The skin of the pig closely resembles human skin and contains clonogenic keratinocytes that can be serially cultivated, cloned or transduced with a gene encoding GFP (Green Fluorescent Protein) by means of recombinant retroviral vectors. Cultured epidermal autografts can be successfully transplanted and their behavior recapitulate our observations in the human. Our experiments confirm that the number of epidermal stem cells rapidly decreases following transplantation. Most importantly, the regenerated epithelium contains dividing cells but little apoptotic cells, thus indicating that transplanted stem cells are pushed toward differentiation in response to the transplantation procedure. Conclusions: The minipig model is extremely useful to investigate stem cell fate during transplantation in human. Understanding engraftment is crucial to improve cell therapy and to design a more efficient generation of epidermal stem cell based products

Endoscopic epiglottopexy using Lichtenberger's needle carrier to avoid breakdown of repair

Severe type III laryngomalacia LM is represented by a retroflexed epiglottis that touches the posterior pharyngeal wall and obstructs the laryngeal inlet. Endoscopic epiglottopexy is advised in such cases wherein pexy sutures are passed between the epiglottis and base of tongue. Using conventional needle carriers, it is difficult to pass such sutures that go deep enough into the tongue base. Such a pexy is prone to a break down. We describe a novel technique of placing these glossoepiglottic sutures using the Lichtenberger's needle carrier. We used this technique in three patients with excellent results and report no complications. We propose to use this technique in cases of epiglottic prolapse seen in severe LM and certain hypotonic conditions

Human clonal urothelial cells arising from single ureteral cell.

<p>(A) Schematics showing the passage from the single selected cell to the in vivo implantated clonal cell pellet. (B,C and D) Human urothelial holoclone, meroclone and paraclone cultures arising from one single ureteral cell. (E and F) Growth curve of a human urothelial holoclone (G) <i>In vivo</i> urothelial differentiation of human ureteral urothelial holoclone pellets implanted into the subcapsular space of the Swiss nu/nu mice, expressing cytokeratin 7, uroplakin-2 and uroplakin 3 (scale bars, 10 µm). Note the “micro-bladder” like structure.</p

Urothelial cell differentiation and “micro-bladder” formation in mice.

<p>(A and B) Hematoxylin & eosin (H&E) staining of an implanted mass-cultured, native porcine bladder urothelial cell pellet into the subcapsular space of a Swiss nu/nu mice kidney (A: scale bar 500 µm, B: scale bar 50 µm, urothelial bundle-like structure indicated with black star and epithelial “micro-bladder” like structure indicated with red star). (C–H) Immunohistochemistry of the implanted urothelial pellet forming urothelial bundle-like structures (C, E and G) and “micro-bladder” like structures (D, F and H) using antibodies against uroplakin-2 (C and D), uroplakin-3 (E and F) and Ki-67 (G and H) (scale bars, 10 µm).</p

Clonal analysis of porcine urethral cells.

<p>(CA = clone area, Estim CN = estimated cell number, GT = generation time, GN = generation number, GC = growing colony, nb = number, AB = aborted).</p

Porcine clonal urothelial cells arising from a single urethral cell.

<p>(A, B and C) Porcine urethelial holoclone, meroclone and paraclone cultures arising from a single urethral cell. (D and E) Growth curves of a porcine urothelial holoclone. (F, G) <i>In vivo</i> urothelial differentiation of porcine urethral urothelial holoclonal cell pellets implanted into the subcapsular space of the Swiss nu/nu mice, expressing cytokeratin 7, uroplakin-2 and uroplakin 3 (scale bars, 10 µm). Note the “micro-bladder” like structure.</p

p63 expression in clonal human urothelial cells and urothelium.

<p>(A) p63-expression in a human urothelial holoclone culture at passage 2. (B) p63-expression in a human urothelial holoclone culture at passage 5. (C) p63-expression in a human urothelial meroclone culture at passage 2. (D) p63-expression in a human ureter biopsy (scale bars, 10 µm). Note the DAPI positive together with GFP negative cells are 3T3-J2 cells and DAPI positive together with GFP positive cells are urothelial cells.</p

Clonal analysis of human ureteral cells.

<p>(CA = clone area, Estim CN = estimated cell number, GT = generation time, GN = generation number, GC = growing colony, nb = number, AB = aborted).</p

Clonal analysis of porcine bladder cells.

<p>(CA = clone area, Estim CN = estimated cell number, GT = generation time, GN = generation number, GC = growing colony, nb = number, AB = aborted).</p

Wet adhesive hydrogels to correct malacic trachea (tracheomalacia) A proof of concept

Summary: Tracheomalacia (TM) is a condition characterized by a weak tracheal cartilage and/or muscle, resulting in excessive collapse of the airway in the newborns. Current treatments including tracheal reconstruction, tracheoplasty, endo- and extra-luminal stents have limitations. To address these limitations, this work proposes a new strategy by wrapping an adhesive hydrogel patch around a malacic trachea. Through a numerical model, first it was demonstrated that a hydrogel patch with sufficient mechanical and adhesion strength can preserve the trachea’s physiological shape. Accordingly, a new hydrogel providing robust adhesion on wet tracheal surfaces was synthesized employing the hydroxyethyl acrylamide (HEAam) and polyethylene glycol methacrylate (PEGDMA) as main polymer network and crosslinker, respectively. Ex vivo experiments revealed that the adhesive hydrogel patches can restrain the collapsing of malacic trachea under negative pressure. This study may open the possibility of using an adhesive hydrogel as a new approach in the difficult clinical situation of tracheomalacia