Infusion of Some but Not All Types of Human Perinatal Stromal Cells Prevent Organ Fibrosis in a Humanized Graft versus Host Disease Murine Model.

Allogeneic transplant rejection represents a medical complication that leads to high morbidity and mortality rates. There are no treatments to effectively prevent fibrosis; however, there is great interest in evaluating the use of perinatal mesenchymal stromal cells (MSCs) and other MSCs to prevent fibrosis associated with chronic rejection. In this study, we isolated human perinatal stromal cells (PSCs) from amnion (AM-PSC), placental villi (PV-PSC), and umbilical cord (UC-PSC) tissues, demonstrating the phenotypic characteristics of MSCs as well as a >70% expression of the immunomodulatory markers CD273 and CD210. The administration of a single dose (250,000 cells) of each type of PSC in a humanized graft versus host disease (hGvHD) NSG® murine model delayed the progression of the disease as displayed by weight loss and GvHD scores ranging at various levels without affecting the hCD3+ population. However, only PV-PSCs demonstrated an increased survival rate of 50% at the end of the study. Furthermore, a histopathological evaluation showed that only PV-PSC cells could reduce human CD45+ cell infiltration and the fibrosis of the lungs and liver. These findings indicate that not all PSCs have similar therapeutic potential, and that PV-PSC as a cell therapeutic may have an advantage for targeting fibrosis related to allograft rejection.

Hepatocyte-like cells derived from human amniotic epithelial, bone marrow, and adipose stromal cells display enhanced functionality when cultured on decellularized liver substrate.

Transplantation of primary hepatocytes has been used in treatments for various liver pathologies and end-stage liver disease. However, shortage of donor tissue and the inability of hepatocyte proliferation in vitro have lead to alternative methods such as stem cell-derived hepatocyte-like cells (HLCs). Mesenchymal stromal/stem cells, and amniotic epithelial cells were isolated from human bone marrow (BM-MSCs), lipoaspirates (ASCs), and amniotic tissue (AECs) respectively. All cells were differentiated into HLCs on plates coated with Type I collagen or Porcine Liver Extracellular Matrix (PLECM-AA) matrix. Flow cytometry of BM-MSCs and ASCs, and AECs showed high expression of MSC-specific and embryonic stem cell markers respectively. All cell types differentiated into osteocytes, chondrocytes, and adipocytes. All cell type-derived HLCs presented the typical cuboidal primary hepatocyte morphology on PLECM-AA and fewer vacuoles (AECs) compared to HLCs cultured on type I collagen. Gene analysis of all cell type-derived HLCs cultured on PLECM-AA revealed higher upregulation of genes involved in drug transportation and metabolism compared to HLCs cultured on type I collagen. Although, HLCs cultured on PLECM-AA displayed some hepatocyte-related function and bioactivity, overall gene expression was lower compared to that of primary hepatocytes suggesting that caution should be taken when considering using HLCs to replace total hepatocyte functionality.

Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture: Method Optimization and Comparison.

Biologic substrates, prepared by decellularizing and solubilizing tissues, have been of great interest in the tissue engineering field because of the preservation of complex biochemical constituents found in the native extracellular matrix (ECM). The integrity of the ECM is critical for cell behavior, adhesion, migration, differentiation, and proliferation that in turn affect homeostasis and tissue regeneration. Previous studies have shown that various processing methods have a distinctive way of affecting the composition of the decellularized ECM. In this study, we developed a bioactive substrate for hepatocytes in vitro, made of decellularized and solubilized liver tissue. The present work is a comparative approach of 2 different methods. First, we decellularized porcine liver tissue with ammonium hydroxide versus a sodium deoxycholate method, then characterized the decellularized tissue using various methods including double stranded DNA (dsDNA) content, DNA size, immunogenicity, and mass spectrometry. Second, we solubilized the decellularized porcine liver with hydrochloric acid versus acetic acid (AA) and characterized the resultant solubilized tissues using relevant methodologies including protein yield, immunogenicity, and bioactivity. Finally, we isolated primary porcine hepatocytes, cultured, and evaluated their bioactivity on the optimized decellularized-solubilized liver substrate. The decellularized porcine liver ECM processed by the ammonium hydroxide method and solubilized with AA displayed higher ECM integrity, low dsDNA, no evidence of intact nuclei, low human monocyte chemoattraction, and the presence of key molecules typically found in the native liver, a very important element for normal cell function. In addition, primary porcine hepatocytes showed enhanced functionality including albumin and urea production and bile canaliculi formation when cultured on the developed liver substrate compared to type I collagen.

C-terminal fragment of transforming growth factor beta-induced protein (TGFBIp) is required for apoptosis in human osteosarcoma cells.

Transforming growth factor beta-induced protein (TGFBIp), is secreted into the extracellular space. When fragmentation of C-terminal portions is blocked, apoptosis is low, even when the protein is overexpressed. If fragmentation occurs, apoptosis is observed. Whether full-length TGFBIp or integrin-binding fragments released from its C-terminus is necessary for apoptosis remains equivocal. More importantly, the exact portion of the C-terminus that conveys the pro-apoptotic property of TGFBIp is uncertain. It is reportedly within the final 166 amino acids. We sought to determine if this property is dependent upon the final 69 amino acids containing the integrin-binding, EPDIM and RGD, sequences. With MG-63 osteosarcoma cells, transforming growth factor (TGF)-beta1 treatment increased expression of TGFBIp over 72 h (p<0.001). At this time point, apoptosis was significantly increased (p<0.001) and was prevented by an anti-TGFBIp, polyclonal antibody (p<0.05). Overexpression of TGFBIp by transient transfection produced a 2-fold increase in apoptosis (p<0.01). Exogenous purified TGFBIp at concentrations of 37-150 nM produced a dose dependent increase in apoptosis (p<0.001). Mass spectrometry analysis of TGFBIp isolated from conditioned medium of cells treated with TGF-beta1 revealed truncated forms of TGFBIp that lacked integrin-binding sequences in the C-terminus. Recombinant TGFBIp truncated, similarly, at amino acid 614 failed to induce apoptosis. A recombinant fragment encoding the final 69 amino acids of the TGFBIp C-terminus produced significant apoptosis. This apoptosis level was comparable to that induced by TGF-beta1 upregulation of endogenous TGFBIp. Mutation of the integrin-binding sequence EPDIM, but not RGD, blocked apoptosis (p<0.001). These pro-apoptotic actions are dependent on the C-terminus most likely to interact with integrins.