Marién Cobo

CD105 (Endoglin)-Negative Murine Mesenchymal Stromal Cells Define a New Multipotent Subpopulation with Distinct Differentiation and Immunomodulatory Capacities

Administration of in vitro expanded mesenchymal stromal cells (MSCs) represents a promising therapy for regenerative medicine and autoimmunity. Both mouse and human MSCs ameliorate autoimmune disease in syn-, allo- and xenogeneic settings. However, MSC preparations are heterogeneous which impairs their therapeutic efficacy and endorses variability between experiments. This heterogeneity has also been a main hurdle in translating experimental MSC data from mouse models to human patients. The objective of the present manuscript has been to further characterize murine MSCs (mMSCs) with the aim of designing more efficient and specific MSC-based therapies. We have found that mMSCs are heterogeneous for endoglin (CD105) expression and that this heterogeneity is not due to different stages of MSC differentiation. CD105 is induced on a subpopulation of mMSCs early upon in vitro culture giving rise to CD105+ and CD105- MSCs. CD105+ and CD105- mMSCs represent independent subpopulations that maintain their properties upon several passages. CD105 expression on CD105+ mMSCs was affected by passage number and cell confluency while CD105- mMSCs remained negative. The CD105+ and CD105- mMSC subpopulations had similar growth potential and expressed almost identical mMSC markers (CD29+CD44+Sca1 + MHC-I+ and CD45-CD11b-CD31-) but varied in their differentiation and immunoregulatory properties. Interestingly, CD105- mMSCs were more prone to differentiate into adipocytes and osteocytes and suppressed the proliferation of CD4+ T cells more efficiently compared to CD105+ mMSCs. Based on these studies we propose to redefine the phenotype of mMSCs based on CD105 expression.

Physiological and tissue-specific vectors for treatment of inherited diseases

After more than 1500 gene therapy clinical trials in the past two decades, the overall conclusion is that for gene therapy (GT) to be successful, the vector systems must still be improved in terms of delivery, expression and safety. The recent development of more efficient and stable vector systems has created great expectations for the future of GT. Impressive results were obtained in three primary immunodeficiencies and other inherited diseases such as congenital blindness, adrenoleukodystrophy or junctional epidermolysis bullosa. However, the development of leukemia in five children included in the GT clinical trials for X-linked severe combined immunodeficiency and the silencing of the therapeutic gene in the chronic granulomatous disease clearly showed the importance of improving safety and efficiency. In this review, we focus on the main strategies available to achieve physiological or tissue-specific expression of therapeutic transgenes and discuss the importance of controlling transgene expression to improve safety. We propose that tissue-specific and/or physiological viral vectors offer the best balance between efficiency and safety and will be the tools of choice for future clinical trials in GT of inherited diseases.

Hematopoietic-specific lentiviral vectors circumvent cellular toxicity due to ectopic expression of Wiskott-Aldrich syndrome protein.

Efficient and safe gene modification of hematopoietic stem cells is a requirement for gene therapy of primary immunodeficiencies such as Wiskott-Aldrich syndrome. However, deregulated expression or ectopic expression in the progeny of transduced nonhematopoietic progenitor cells may lead to unwanted toxicity. We therefore analyzed the effect of ectopic expression of Wiskott-Aldrich syndrome protein (WASp) and the potential benefits of hematopoietic-specific lentiviral vectors (driven by the WAS proximal promoter). Overexpression of WASp by constitutive lentiviral vectors is highly toxic in nonhematopoietic cells because it causes dramatic changes in actin localization and polymerization that result in decreased cell viability, as evidenced by a significant growth disadvantage of WASp-overexpressing nonhematopoietic cells and increased cell death. These toxic effects do not affect cells of hematopoietic origin because, remarkably, we found that WASp cannot be readily overexpressed in T cells, even after multiple vector integrations per cell. The adverse cellular effects found after transduction of nonhematopoietic cells with constitutive lentiviral vectors are overcome by the use of transcriptionally targeted lentiviral vectors expressing WASp, which, at the same time, are efficient tools for gene therapy of WAS as demonstrated by their ability to reconstitute cellular defects from WASp-deficient mouse and human cells. We therefore postulate that transcriptionally regulated lentiviral vectors represent a safer and efficient alternative for the development of clinical protocols of WAS gene therapy.

Mesenchymal Stem Cells Expressing Vasoactive Intestinal Peptide Ameliorate Symptoms in a Model of Chronic Multiple Sclerosis

Multiple sclerosis (MS) is a severe debilitating disorder characterized by progressive demyelination and axonal damage of the central nervous system (CNS). Current therapies for MS inhibit the immune response and demonstrate reasonable benefits if applied during the early phase of relapsing–remitting MS (RRMS) while there are no treatments for patients that progress neither to the chronic phase nor for the primary progressive form of the disease. In this manuscript, we have studied the therapeutic efficacy of a cell and gene therapy strategy for the treatment of a mouse model of chronic MS [myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE)]. We used allogenic mesenchymal stem cells (MSCs) as a therapeutic tool and also as vehicle to deliver fully processed 3.3-kDa vasoactive intestinal peptide (VIP) to the peripheral immune organs and to the inflamed CNS. Intraperitoneal administrations of MSCs expressing VIP stopped progression and reduced symptoms when administered at peak of disease. The improvement in clinical score correlated with diminished peripheral T-cell responses against MOG as well as lower inflammation, lower demyelination, and higher neuronal integrity in the CNS. Interestingly, neither lentiviral vectors expressing VIP nor unmodified MSCs were therapeutic when administer at the peak of disease. The increased therapeutic effect of MSCs expressing VIP over unmodified MSCs requires the immunoregulatory and neuroprotective roles of both VIP and MSCs and the ability of the MSCs to migrate to peripheral lymph organs and the inflamed CNS.

Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines

Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and gene therapy approaches. Combining LVs with drug-inducible expression systems should allow tight control of transgene expression with minimal side effect on relevant target cells. A new doxycycline-regulated system based on the original TetR repressor was developed in 1998 as an alternative to the TetR-VP16 chimeras (tTA and rtTA) to avoid secondary effects due to the expression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscript we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have also showed that this dual vector system can generate regulatable bulk cell lines (expressing high levels of TetR) that are able to modulate transgene expression either by varying doxycycline concentration and/or by varying the amount of CTetOE vector genomes per cell. Based on these results we have developed a new all-in-one lentiviral vector (CEST) driving the expression of TetR through the SFFV promoter and the expression of eGFP through the doxycycline-responsive CMV-TetO operon. This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) cells. Bulk doxycycline-responsive cell lines express high levels of the transgene with low amount of doxycycline and are phenotypically indistinct from its parental cells.

Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity

Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)‐β1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency‐associated peptide (LAP)/TGF‐β1 to the cell surface of activated Foxp3+ regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF‐β1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF‐β1 and reduced their proliferative capacity in a TGF‐β1‐independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T‐cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker. Stem Cells 2015;33:183–195

A tissue-specific, activation-inducible, lentiviral vector regulated by human CD40L proximal promoter sequences

The application of new protocols for gene therapy against monogenic diseases requires the development of safer therapeutic vectors, particularly in the case of diseases in which expression of the mutated gene is subject to fine regulation, as it is with CD40L (CD154). CD40L, the gene mutated in the X-linked hyper-immunoglobulin M syndrome (HIGM1), is tightly regulated to allow surface expression of its product only on T cells stimulated by antigen encounter. Previous studies in an HIGM1 animal model showed that transduction of progenitor cells corrected the syndrome but caused a thymic lymphoproliferative disease because of the unregulated expression of the transgene by constitutive vectors. To develop a tissue-specific, activation-inducible, lentiviral vector (LV) for gene therapy to counter HIGM1, we have constructed two self-inactivating LVs, pCD40L-eGFP and pCD40L-CD40L, regulated by a 1.3 kb fragment of the human CD40L proximal promoter. The expression of pCD40L-eGFP LV is restricted to cells in which mRNA transcripts of the endogenous CD40L gene can be detected. Moreover, the expression of the reporter gene in primary T lymphocytes depends on the activation state of the cells. Remarkably, primary HIGM1 lymphocytes transduced with pCD40L-CD40L LV expressed CD40L only after T-cell stimulation. Therefore, the CD40L-promoter-driven vectors are able to achieve a near-physiological expression pattern that follows very closely that of the endogenous CD40L gene.

Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome

SUMMARY Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS), which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK) development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN) pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells) resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41ɑ, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS patients and as a cellular model to study new therapeutic strategies.

SCL/TAL1-mediated Transcriptional Network Enhances Megakaryocytic Specification of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.

A Chimeric HS4-SAR Insulator (IS2) That Prevents Silencing and Enhances Expression of Lentiviral Vectors in Pluripotent Stem Cells

Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3′ LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells.