Long-Term Hematopoietic Engraftment of Congenic Amniotic Fluid Stem Cells after in Utero Intraperitoneal Transplantation to Immune Competent Mice

Clinical success of in utero transplantation (IUT) using allogeneic hematopoietic stem cells (HSCs) has been limited to fetuses that lack an immune response to allogeneic cells due to severe immunological defects, and where transplanted genetically normal cells have a proliferative or survival advantage. Amniotic fluid (AF) is an autologous source of stem cells with hematopoietic potential that could be used to treat congenital blood disorders. We compared the ability of congenic and allogeneic mouse AF stem cells (AFSC) to engraft the hematopoietic system of time-mated C57BL/6J mice (E13.5). At 4 and 16 weeks of age, multilineage donor engraftment was higher in congenic versus allogeneic animals. In vitro mixed lymphocyte reaction confirmed an immune response in the allogeneic group with higher CD4 and CD8 cell counts and increased proliferation of stimulated lymphocytes. IUT with congenic cells resulted in 100% of donor animals having chimerism of around 8% and successful hematopoietic long-term engraftment in immune-competent mice when compared with IUT with allogeneic cells. AFSCs may be useful for autologous cell/gene therapy approaches in fetuses diagnosed with congenital hematopoietic disorders

Isolation of esophageal stem cells with potential for therapy

PURPOSE: Long-gap esophageal atresia represents a significant challenge for pediatric surgeons and current surgical approaches are associated with significant morbidity. A tissue-engineered esophagus, comprising cells seeded onto a scaffold, represents a therapeutic alternative. In this study, we aimed to determine the optimal techniques for isolation and culture of mouse esophageal epithelial cells and to isolate CD34-positive esophageal epithelial stem cells from cadaveric mouse specimens. METHODS: Primary epithelial cells were isolated from mouse esophagi by enzymatic dissociation from the mucosal layer (Dispase, Trypsin/EDTA) using three different protocols. In protocol A, isolated mucosa was minced and incubated with trypsin once. In protocol B, intact mucosal sheets underwent two trypsin incubations yielding a single-cell suspension. In protocol C, intact mucosa explants were plated epithelial side down. Epithelial cells were cultured on collagen-coated wells. RESULTS: Initial findings showed that Protocol B gave the best results in terms of yield, viability, and least contamination with different cell types and microbes. Esophageal epithelial cells isolated using Protocol B were stained for CD34 and sorted using fluorescence-activated cell sorting (FACS). Of the total cells sorted, 8.3 % (2–11.3) [%median (range)] were CD34 positive. CONCLUSIONS: Our results demonstrate that mouse esophageal epithelial cells can be successfully isolated from fresh mouse esophagi using two consecutive trypsin incubations of intact mucosal sheets. Furthermore, the cells obtained using this method were successfully stained for CD34, a putative esophageal epithelial stem cell marker. Further research into the factors necessary for the successful proliferation of CD34 positive stem cell lines is needed to progress toward clinical application

Long-term cryopreservation of decellularised oesophagi for tissue engineering clinical application

textabstractOesophageal tissue engineering is a therapeutic alternative when oesophageal replacement is required. Decellularised scaffolds are ideal as they are derived from tissue-specific extracellular matrix and are non-immunogenic. However, appropriate preservation may significantly affect scaffold behaviour. Here we aim to prove that an effective method for shortand long-term preservation can be applied to tissue engineered products allowing their translation to clinical application. Rabbit oesophagi were decellularised using the detergentenzymatic treatment (DET), a combination of deionised water, sodium deoxycholate and DNase-I. Samples were stored in phosphate-buffered saline solution at 4°C (4°C) or slow cooled in medium with 10% Me2SO at -1°C/min followed by storage in liquid nitrogen (SCM). Structural and functional analyses were performed prior to and after 2 and 4 weeks and 3 and 6 months of storage under each condition. Efficient decellularisation was achieved after 2 cycles of DET as determined with histology and DNA quantification, with preservation of the ECM. Only the SCM method, commonly used for cell storage, maintained the architecture and biomechanical properties of the scaffold up to 6 months. On the contrary, 4°C method was effective for short-term storage but led to a progressive distortion and degradation of the tissue architecture at the following time points. Efficient storage allows a timely use of decellularised oesophagi, essential for clinical translation. Here we describe that slow cooling with cryoprotectant solution in liquid nitrogen vapour leads to reliable long-term storage of decellularised oesophageal scaffolds for tissue engineering purposes

Preservation of micro-architecture and angiogenic potential in a pulmonary acellular matrix obtained using intermittent intra-tracheal flow of detergent enzymatic treatment

Tissue engineering of autologous lung tissue aims to become a therapeutic alternative to transplantation. Efforts published so far in creating scaffolds have used harsh decellularization techniques that damage the extracellular matrix (ECM), deplete its components and take up to 5 weeks to perform. The aim of this study was to create a lung natural acellular scaffold using a method that will reduce the time of production and better preserve scaffold architecture and ECM components. Decellularization of rat lungs via the intratracheal route removed most of the nuclear material when compared to the other entry points. An intermittent inflation approach that mimics lung respiration yielded an acellular scaffold in a shorter time with an improved preservation of pulmonary micro-architecture. Electron microscopy demonstrated the maintenance of an intact alveolar network, with no evidence of collapse or tearing. Pulsatile dye injection via the vasculature indicated an intact capillary network in the scaffold. Morphometry analysis demonstrated a significant increase in alveolar fractional volume, with alveolar size analysis confirming that alveolar dimensions were maintained. Biomechanical testing of the scaffolds indicated an increase in resistance and elastance when compared to fresh lungs. Staining and quantification for ECM components showed a presence of collagen, elastin, GAG and laminin. The intratracheal intermittent decellularization methodology could be translated to sheep lungs, demonstrating a preservation of ECM components, alveolar and vascular architecture. Decellularization treatment and methodology preserves lung architecture and ECM whilst reducing the production time to 3 h. Cell seeding and in vivo experiments are necessary to proceed towards clinical translation

Optimization of Liver Decellularization Maintains Extracellular Matrix Micro-Architecture and Composition Predisposing to Effective Cell Seeding

<div><p>Hepatic tissue engineering using decellularized scaffolds is a potential therapeutic alternative to conventional transplantation. However, scaffolds are usually obtained using decellularization protocols that destroy the extracellular matrix (ECM) and hamper clinical translation. We aim to develop a decellularization technique that reliably maintains hepatic microarchitecture and ECM components. Isolated rat livers were decellularized by detergent-enzymatic technique with (EDTA-DET) or without EDTA (DET). Histology, DNA quantification and proteomics confirmed decellularization with further DNA reduction with the addition of EDTA. Quantification, histology, immunostaining, and proteomics demonstrated preservation of extracellular matrix components in both scaffolds with a higher amount of collagen and glycosaminoglycans in the EDTA-DET scaffold. Scanning electron microscopy and X-ray phase contrast imaging showed microarchitecture preservation, with EDTA-DET scaffolds more tightly packed. DET scaffold seeding with a hepatocellular cell line demonstrated complete repopulation in 14 days, with cells proliferating at that time. Decellularization using DET preserves microarchitecture and extracellular matrix components whilst allowing for cell growth for up to 14 days. Addition of EDTA creates a denser, more compact matrix. Transplantation of the scaffolds and scaling up of the methodology are the next steps for successful hepatic tissue engineering.</p></div

Seeding of the DET scaffolds by microinjection demonstrates complete repopulation at 14 days.

<p>(A) DET scaffolds were microinjected with HepG2 cells in a multifocal manner and the seeded constructs harvested at day 1, 4 and 14. (B) H&E staining demonstrated a wide distribution across the scaffold at day 1 and a complete repopulation at 14 days (B). At higher magnification, cells were seen repopulating the hepatic spaces, forming colonies and secreting their own ECM at 14 days (C, asterisk). Ki67 staining demonstrated proliferation at days 1 and 4, with only a few ki67-positive cells at 14 days (D). Quantification of ki67-positive cells as a percentage to DAPI-positive cells (F). Staining for CC-3 showed a few positive cells across the three time-points (E), with quantification demonstrating a non-significant increase (G); scale bar: 100μm.</p

Addition of EDTA to the protocol makes the matrix more compact in the parenchyma and vasculature.

<p>(A-C) SEM confirmed acellularity in the scaffolds, demonstrating composition by a three-dimensional network of connective tissue fibers arranged in a honeycomb-like manner. The structure of the portal triads was identified clearly at low magnification (asterisk). (C) Scaffolds prepared with EDTA-DET were more tightly packed compared to the DET scaffolds, as the porous structure appeared to be compressed. (D) Quantification of the hepatocyte pockets in the EDTA-DET scaffold showed a reduction to approximately 50% of the size in the DET scaffolds. (E, F) Synchrotron-based x-ray phase contrast imaging corroborated these results, demonstrating a denser scaffold in scaffolds pre-treated with EDTA. (G) Infusion of trypan blue dye via the portal vein showed maintenance of the vascular network architecture. There was no dye leakage through the walls to the surrounding tissue; the dye followed the regular pathway of flow to the IVC. Macroscopically, the DET scaffolds were seen to possess a denser vascular network with the dye diffusing through the vessels more readily. (H) Dye intensity within the DET scaffolds followed an S-shaped curve, reaching a maximum point at approximately 35 seconds. Dye intensity within the EDTA-DET scaffold increased in a less steep S-shaped manner with the exponential part lasting 45 seconds instead of 15. Maximum intensity was reached at 75 seconds. (I) These results were paralleled by the quantification of surface area of dye distribution with the point of maximal surface area coverage having a difference of 40 seconds between the two scaffolds; #: p<0.001, compared to DET scaffold, scale bar: 1cm.</p

Immunostaining demonstrates the preservation of ECM proteins in the decellularized scaffolds.

<p>(A) Collagen I staining in fresh tissue was positive as fine strands in the parenchymal space as well as around the blood vessels (asterisk). This was preserved following decellularization with a strong signal from vascular structures both in DET and EDTA-DET scaffolds. (B, C) Collagen III and collagen IV staining demonstrated a similar distribution and preservation in fresh tissue and decellularized scaffolds. Weak parenchymal staining was observed, and a strong signal surrounding vascular and biliary structures. EDTA-DET scaffolds demonstrated slightly increased staining in the parenchymal space when compared to DET scaffolds. (D) Fibronectin showed strong staining around the main blood vessels in fresh tissue with a more distributed signal pattern in decellularized scaffolds. (E) Laminin showed strong staining around the main blood vessels in fresh tissue with a more distributed signal pattern in decellularized scaffolds; scale bar: 100 μm.</p