Liver Regeneration by Hematopoietic Stem Cells: Have We Reached the End of the Road?

The liver is the organ with the highest regenerative capacity in the human body. However, various insults, including viral infections, alcohol or drug abuse, and metabolic overload, may cause chronic inflammation and fibrosis, leading to irreversible liver dysfunction. Despite advances in surgery and pharmacological treatments, liver diseases remain a leading cause of death worldwide. To address the shortage of donor liver organs for orthotopic liver transplantation, cell therapy in liver disease has emerged as a promising regenerative treatment. Sources include primary hepatocytes or functional hepatocytes generated from the reprogramming of induced pluripotent stem cells (iPSC). Different types of stem cells have also been employed for transplantation to trigger regeneration, including hematopoietic stem cells (HSCs), mesenchymal stromal cells (MSCs), endothelial progenitor cells (EPCs) as well as adult and fetal liver progenitor cells. HSCs, usually defined by the expression of CD34 and CD133, and MSCs, defined by the expression of CD105, CD73, and CD90, are attractive sources due to their autologous nature, ease of isolation and cryopreservation. The present review focuses on the use of bone marrow HSCs for liver regeneration, presenting evidence for an ongoing crosstalk between the hematopoietic and the hepatic system. This relationship commences during embryogenesis when the fetal liver emerges as the crossroads between the two systems converging the presence of different origins of cells (mesoderm and endoderm) in the same organ. Ample evidence indicates that the fetal liver supports the maturation and expansion of HSCs during development but also later on in life. Moreover, the fact that the adult liver remains one of the few sites for extramedullary hematopoiesis-albeit pathological-suggests that this relationship between the two systems is ongoing. Can, however, the hematopoietic system offer similar support to the liver? The majority of clinical studies using hematopoietic cell transplantation in patients with liver disease report favourable observations. The underlying mechanism-whether paracrine, fusion or transdifferentiation or a combination of the three-remains to be confirmed.

The -938C>A Polymorphism in MYD88 Is Associated with Susceptibility to Tuberculosis: A Pilot Study.

Introduction. Tuberculosis (TB) is a major disease worldwide, caused by Mycobacterium tuberculosis (MTB) infection. The Toll-Like Receptor (TLR) pathway plays a crucial role in the recognition of MTB. Aim. The present study aimed to investigate the involvement of myeloid differentiation primary response protein 88 (MYD88) gene polymorphisms in TB. Materials and Methods. A total of 103 TB cases and 92 control subjects were genotyped for the MYD88 -938C>A (rs4988453) and 1944C>G (rs4988457) polymorphisms. Results. The MYD88 -938CA and -938AA genotypes were associated with an increased risk for tuberculosis with odds ratio (OR) of 5.71 (95% confidence intervals [CIs] 2.89-11.28, p = 0.01). Conclusions. The MYD88 -938C>A genetic polymorphism is associated with increased susceptibility to TB and may serve as a marker to screen individuals who are at risk.

Generation of human ß-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with ß-thalassemia (ß-thal) with the aim to generate trangene-free ß-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in ß-thal-iPSC lines. After 10 serial passages in vitro, ß-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free ß-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

A foamy virus vector system for stable and efficient RNAi expression in mammalian cells.

The promise of the RNA interference (RNAi) technology is equally dependent on the efficiency and stability of gene silencing. The aim of the present study was the development of foamy virus (FV) vectors for stable RNAi, utilizing two potent RNA polymerase III (Pol III) promoters. Using green fluorescent protein as a target gene, we examined the efficiency of mouse U6 (mU6) and human H1 Pol III promoters in different human cell lines and mouse hematopoietic stem cells (HSCs) ex vivo and in vivo, following bone marrow transplantation. Both our mU6 and H1 FV vectors mediated very efficient gene silencing with as low as one vector copy per cell. However, transduction of human cell lines with FV vectors expressing short hairpin RNA from mU6 led to the gradual elimination of cells in culture, as opposed to H1-harboring cells, underscoring the importance of the expression system or cellular context in the evaluation of the overall RNAi effects. The efficiency and stability of the H1 vectors were further shown by the successful silencing of BCR-ABL in K562 cells. Accordingly, mU6 vectors induced efficient and stable gene silencing in mouse HSCs following bone marrow transplantation. Our work is the first in vivo study on the efficiency and stability of RNAi gene silencing in HSCs with FV vectors, currently a safe alternative for viral gene transfer.

Genetic correction of X-linked chronic granulomatous disease with novel foamy virus vectors.

OBJECTIVE: The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the CYBB gene encoding gp91(phox), the larger subunit of the oxidase flavocytochrome b(558). Affected individuals suffer from recurrent life-threatening infections due to impaired superoxide production by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Novel foamy virus vectors expressing the human codon-optimized gp91(phox) were evaluated for the genetic correction of the disease in the X-CGD cell line and in X-CGD mouse model. MATERIALS AND METHODS: The vectors were evaluated in vitro, in the human X-CGD PLB-985 cell line and in the X-CGD bone marrow Lin(-) cells. Transplantation of transduced Lin(-) cells was performed in X-CGD mice after busulfan conditioning. Real-time polymerase chain reaction was used for chimerism and vector copy number determination. Restoration of reduced NADPH oxidase production was assessed by nitrobluetetrazolium and dihydrorhodamine assays. RESULTS: High and stable gp91(phox) expression, as well as reconstitution of reduced NADPH activity, was achieved in the human X-CGD PLB-985 cell line and in primary murine X-CGD hematopoietic stem cells ex vivo. Transplantation of transduced bone marrow hematopoietic stem cells in the murine model of X-CGD, even with low multiplicities of infection (MOI), reconstituted the levels of oxidase-producing neutrophils and provided enzymatic activity that reached 70% of normal. CONCLUSIONS: Foamy virus vectors expressing the human gp91(phox) transgene constitute potential candidates for the gene therapy of CGD because they combine lack of pathogenicity with efficacy even at low MOI.