Modeling of hemophilia A using patient-specific induced pluripotent stem cells derived from urine cells

Abstract

Aims

Hemophilia A (HA) is a severe, congenital bleeding disorder caused by the deficiency of clotting factor VIII (FVIII). For years, traditional laboratory animals have been used to study HA and its therapies, although animal models may not entirely mirror the human pathophysiology. Human induced pluripotent stem cells (iPSCs) can undergo unlimited self-renewal and differentiate into all cell types. This study aims to generate hemophilia A (HA) patient-specific iPSCs that differentiate into disease-affected hepatocyte cells. These hepatocytes are potentially useful for in vitro disease modeling and provide an applicable cell source for autologous cell therapy after genetic correction.

Main methods

In this study, we mainly generated iPSCs from urine collected from HA patients with integration-free episomal vectors PEP4-EO2S-ET2K containing human genes OCT4, SOX2, SV40LT and KLF4, and differentiated these iPSCs into hepatocyte-like cells. We further identified the genetic phenotype of the FVIII genes and the FVIII activity in the patient-specific iPSC derived hepatic cells.

Key findings

HA patient-specific iPSCs (HA-iPSCs) exhibited typical pluripotent properties evident by immunostaining, in vitro assays and in vivo assays. Importantly, we showed that HA-iPSCs could differentiate into functional hepatocyte-like cells and the HA-iPSC-derived hepatocytes failed to produce FVIII, but otherwise functioned normally, recapitulating the phenotype of HA disease in vitro.

Significance

HA-iPSCs, particular those generated from the urine using a non-viral approach, provide an efficient way for modeling HA in vitro. Furthermore, HA-iPSCs and their derivatives serve as an invaluable cell source that can be used for gene and cell therapy in regenerative medicine.

Introduction

Hemophilia A (HA), an X-chromosome-linked, recessive disorder occurring in 1 or 2 individuals per 10,000, is a severe, congenital disease caused by the deficiency of factor VIII (FVIII) (Bolton-Maggs and Pasi, 2003, Roth et al., 2001, Xu et al., 2009). A variety of FVIII mutations in HA have been described, including point mutations, deletions, insertions, and rearrangements/inversions (Tuddenham et al., 1991). The complexity of the mutations gives rise to a variety of difficulties when correcting the genetic defects. Clinically, severe HA (< 1 % factor activity) can result in disability and death because of spontaneous and prolonged bleeding (Bolton-Maggs and Pasi, 2003). Notably, 40–50 % of patients with severe HA disease have an inversion involving intron 22 of the FVIII gene (Bowen, 2002). Currently, HA can be treated with fixed-dose FVIII prophylaxis or factor replacement therapies (Dielis et al., 2008). However, the two treatments have limitations in their efficacy, cost, availability, and side effects including the development of neutralizing antibodies (Shi et al., 2008). These limitations necessitate the development of new drugs and new treatments and further elucidation of the mechanisms of HA. To this end, animal models have contributed much to the understanding of HA pathophysiology and to HA drug screening. However, data obtained from animals cannot be applied directly to humans, and drugs that are effective in animal models often fail in humans because animal systems do not faithfully mirror the biochemical, physiologic, anatomic and genetic characteristics in humans (Saha and Jaenisch, 2009).

In 2006, Yamanaka established a method to generate induced pluripotent stem cells (iPSCs), paving a new way for modeling relevant human diseases and increasing the promise of gene and cell therapies. Yamanaka's technique was first applied to the study of HA by Xu and colleagues. They induced iPSCs derived from murine tail-tip fibroblasts to differentiate into endothelial progenitor cells expressing FVIII (Xu et al., 2009). Because hepatocytes are the major FVIII-producing cells (Bontempo et al., 1987), researchers further engrafted the endothelial progenitor cells into the livers of mice with hemophilia, which showed sustained FVIII production and the phenotypic correction of the bleeding disorder (Alipio et al., 2010, Xu et al., 2009). This finding suggested that liver transplantation could restore FVIII levels in patients with HA (Bontempo et al., 1987). However, patient-specific iPSCs were not yet available for studies of HA treatment.

In this study, we established a new human HA model using hepatocyte-like cells differentiated from integration-free iPSCs. The integration-free iPSCs were generated from cells from the urine of HA patients with an intron 22 inversion in their FVIII gene. The hepatocyte-like cells derived from the patient-specific iPSCs displayed an intron 22 inversion in the FVIII gene, and failed to secrete FVIII. Our findings provide, not only a good model for studying HA disease but also a cell source for cell therapy after correcting HA mutations with iPSC gene targeting.