In the present study we present a step by

In the present study, we present a step-by-step strategy by which gene-corrected osteopetrotic iPSCs were used to generate hematopoietic progenitor lgk974 able to give rise in vitro to functional osteoclasts, thus providing a proof of principle for an autologous cell therapy approach to treat osteopetrosis and potentially other genetic blood disorders (Hanna et al., 2007).
Results
Discussion Autologous HSC gene therapy represents an emerging therapeutic opportunity for genetic diseases with hematological origin to avoid the requirement of compatible donors. However, viral vector-mediated gene correction of patient HSCs carry the risk of insertional mutagenesis (Nienhuis, 2013) and unregulated transgene expression. Gene correction by homologous recombination would represent a major step forward; however, the difficulty of ex vivo expanding and/or selecting a small fraction of gene-edited HSCs still represents a challenge. As an alternative to ex vivo HSCs, iPSCs represent an innovative source of donor cells. They are pluripotent stem cells derived from the reprogramming of somatic cells, so they have an easily accessible cell source, obtainable without invasive procedures. The feasibility to perform gene targeting is greater, for their propensity to homologous recombination and for the possibility of selecting and expanding clones where gene editing has occurred. In the present study, we tested the feasibility of gene-correction technology using as donor a BAC engineered vector. Taking advantage of the large genomic region carried by this vector, the full-length form of Tcirg1 was inserted, correcting the large mutation that causes the phenotype in the mouse. Since in human ARO the TCIRG1 gene is mainly affected by point mutations located in different positions within the gene locus, the use of a BAC allows the utilization of the same donor vector for every type of mutations found in TCIRG1-dependent patients. In contrast, targeted genome editing using artificial nucleases would imply designing either a different donor template for each mutation, or a single cDNA template, thus not completely restoring the native locus including introns and regulatory sequences. We generated iPSCs using a third-generation self-inactivating excisable lentiviral vector, which proved to be efficacious for obtaining vector-free pluripotent stem cells. More recent techniques, such as the use of the non-integrating Sendai virus, would allow production of iPSCs without all the in vitro manipulations required for Cre-recombinase excision, which include extensive sub-cloning and selection of vector-free and normal karyotype clones. All these procedures could be skipped thus avoiding the risk of introducing additional mutations. Importantly, if pre-natal tissues are used to generate patient-specific iPSCs, as proposed by others (Anchan et al., 2011), autologous iPSC-mediated cell therapy for ARO could be envisioned to treat patients at birth, if not earlier, to prevent bone as well as secondary neurological defects, which cannot be rescued if transplantation is not performed soon after birth. Moreover, iPSCs represent an unlimited supply of corrected cells for additional future use. To evaluate the ability of WT and oc/oc corrected iPSCs to form functional osteoclasts, a protocol for the hematopoietic differentiation was set up, to obtain myeloid cells as well as HSCs/early progenitors. Various differentiation protocols have been described to obtain in vitro the hematopoietic lineage, but the only ones which provide long-term stem cells utilize the overexpression of additional transcription factors, such as Hoxb4, Lhx2, or the combination of Gata2, Gfi1b, cFos, and Etv6 (Hanna et al., 2007, 2010; Takahashi and Yamanaka, 2006). The overexpression of these factors represents an additional manipulation and requires the use of integrating vectors for the stable expression of transgenes. For this reason, we opted to avoid the overexpression of supplementary transgenes, thus inducing differentiation in more physiological conditions. Indeed, in the presence of a hematopoietic cytokine cocktail the EB method induces first the mesoderm development at the expense of the other two germ layers, and then pushes toward the hematopoietic specification, likely through a hemogenic endothelium intermediate. All tested clones behaved similarly in terms of hematopoietic differentiation, with the exception of a slight delay in the kinetics of myeloid differentiation in oc/oc clones that was restored upon gene correction. Despite all the efforts, the scientific community has not yet reached the goal of reproducing in vitro the complex microenvironment made of cells, cytokines, and signals that sustains the correct HSC development. Accordingly, we were not able to demonstrate engraftment of our iPSC-derived hematopoietic progenitors in oc/oc recipient mice (F. Ficara, unpublished data). Further amelioration of the in vitro procedures aimed at obtaining a sufficient number of safe, functional HSCs in a timely manner from somatic cells going through a pluripotent intermediate is therefore needed before iPSC research can be translated in the clinical practice for the cure of diseases with hematological origin. Nevertheless, upon gene correction, oc/oc iPSCs differentiated into hematopoietic progenitors and then to mature osteoclasts able to efficiently resorb dentine in vitro, demonstrating their fully functional correction. Interestingly, it has been proposed that ARO infants could benefit from early transplantation of myeloid progenitors differentiated toward the osteoclast lineage (Cappariello et al., 2010). In the mouse, this approach has been shown to prevent defects in the formation of foramina, which the optic and acoustic nerves pass through, thus reducing visual and hearing defects. In this regard, autologous iPSCs could provide an unlimited source of autologous mature osteoclasts for repeated infusions.