Microvesicles Secreted by Nitric Oxide-Primed Mesenchymal Stromal Cells Boost the Engraftment Potential of Hematopoietic Stem Cells.

Patients with leukemia, lymphoma, severe aplastic anemia, etc. are frequently the targets of bone marrow transplantation, the success of which critically depends on efficient engraftment by transplanted hematopoietic cells (HSCs). Ex vivo manipulation of HSCs to improve their engraftment ability becomes necessary when the number or quality of donor HSCs is a limiting factor. Due to their hematopoiesis-supportive ability, bone marrow-derived mesenchymal stromal cells (MSCs) have been traditionally used as feeder layers for ex vivo expansion of HSCs. MSCs form a special HSC-niche in vivo, implying that signaling mechanisms operative in them would affect HSC fate. We have recently demonstrated that AKT signaling prevailing in the MSCs affect the HSC functionality. Here we show that MSCs primed with nitric oxide donor, Sodium nitroprusside (SNP), significantly boost the engraftment potential of the HSCs co-cultured with them via intercellular transfer of microvesicles (MVs) harboring mRNAs encoding HSC-supportive genes. Our data suggest that these MVs could be used as HSC-priming agents to improve transplantation efficacy. Since both, nitric oxide donors and MSCs are already in clinical use; their application in clinical settings may be relatively straight forward. This approach could also be applied in regenerative medicine protocols. Stem Cells 2019;37:128-138.

AKT Signaling Prevailing in Mesenchymal Stromal Cells Modulates the Functionality of Hematopoietic Stem Cells via Intercellular Communication.

The AKT pathway plays an important role in various aspects of stem cell biology. However, the consequences of constitutive activation of AKT in mesenchymal stromal cells (MSCs) on the fate of hematopoietic stem cells (HSCs) were unknown. Here, we show that bone marrow-derived MSCs expressing a constitutively active AKT1 expand HSCs, but severely affect their functionality. Conversely, stromal cells with silenced AKT1 limit HSC proliferation, but boost their functionality. These effects were related to differential modulation of several important regulatory genes, in both, the cocultured HSCs and in the stromal cells themselves. The detrimental effect of stromal cells with constitutively activated AKT1 involved dynamin-dependent endocytosis, whereas the salutary effect of stromal cells devoid of AKT1 was mediated via GAP junctions. Constitutive activation of AKT1 led to deregulated formation of GAP junctions in the stromal cells, which consequently exhibited strikingly increased intercellular transfer of molecular cargo to the HSCs. Conversely, stromal cells with silenced AKT1 exhibited normal intercellular arrangement of GAP junctions at appositional membrane areas, and did not show aberrant intercellular transfer. Micro-vesicles isolated from conditioned media of the stromal cells not only mimicked the effect of these cells, but also showed stronger effects. This is perhaps the first report demonstrating that AKT1 signaling prevailing in the MSCs regulates HSC functionality through various intercellular communication mechanisms. These findings could have important implications in the use of MSCs in regenerative medicine. Stem Cells 2016;34:2354-2367.

T-Cell Progenitors As A New Immunotherapy to Bypass Hurdles of Allogeneic Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of preference for numerous malignant and non-malignant hemopathies. The outcome of this approach is significantly hampered by not only graft-versus-host disease (GvHD), but also infections and relapses that may occur because of persistent T-cell immunodeficiency following transplantation. Reconstitution of a functional T-cell repertoire can take more than 1 year. Thus, the major challenge in the management of allogeneic HSCT relies on the possibility of shortening the window of immune deficiency through the acceleration of T-cell recovery, with diverse, self-tolerant, and naïve T cells resulting from de novo thymopoiesis from the donor cells. In this context, adoptive transfer of cell populations that can give rise to mature T cells faster than HSCs while maintaining a safety profile compatible with clinical use is of major interest. In this review, we summarize current advances in the characterization of thymus seeding progenitors, and their ex vivo generated counterparts, T-cell progenitors. Transplantation of the latter has been identified as a worthwhile approach to shorten the period of immune deficiency in patients following allogeneic HSCT, and to fulfill the clinical objective of reducing morbimortality due to infections and relapses. We further discuss current opportunities for T-cell progenitor-based therapy manufacturing, including iPSC cell sources and off-the-shelf strategies. These opportunities will be analyzed in the light of results from ongoing clinical studies involving T-cell progenitors.

A DL-4- and TNFα-based culture system to generate high numbers of nonmodified or genetically modified immunotherapeutic human T-lymphoid progenitors.

Several obstacles to the production, expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy. In the context of HSCT, delayed naïve T-cell recovery contributes to poor outcomes. A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human T-lymphoid progenitors (HTLPs), allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus. However, it is challenging to produce HTLPs in the high numbers required to meet clinical needs. Here, we found that adding tumor necrosis factor alpha (TNFα) to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival. This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy (including CAR T-cell therapy).

Ex vivo generated human T-lymphoid progenitors as a tool to accelerate immune reconstitution after partially HLA compatible hematopoietic stem cell transplantation or after gene therapy.

Prolonged T-cell immunodeficiency following HLA- incompatible hematopoietic stem cell transplantation (HSCT) represents a major obstacle hampering the more widespread use of this approach. Strategies to fasten T-cell reconstitution in this setting are highly warranted as opportunistic infections and an increased risk of relapse account for high rates of morbidity and mortality especially during early month following this type of HSCT. We have implemented a feeder free cell system based on the use of the notch ligand DL4 and cytokines allowing for the in vitro differentiation of human T-Lymphoid Progenitor cells (HTLPs) from various sources of CD34+ hematopoietic stem and precursor cells (HSPCs). Co- transplantion of human T-lymphoid progenitors (HTLPs) and non- manipulated HSPCs into immunodeficient mice successfully accelerated the reconstitution of a polyclonal T-cell repertoire. This review summarizes preclinical data on the use of T-cell progenitors for treatment of post- transplantation immunodeficiency and gives insights into the development of GMP based protocols for potential clinical applications including gene therapy approaches. Future clinical trials implementing this protocol will aim at the acceleration of immune reconstitution in different clinical settings such as SCID and leukemia patients undergoing allogeneic transplantation. Apart from pure cell-therapy approaches, the combination of DL-4 culture with gene transduction protocols will open new perspectives in terms of gene therapy applications for primary immunodeficiencies.

Baboon envelope LVs efficiently transduced human adult, fetal, and progenitor T cells and corrected SCID-X1 T-cell deficiency.

T cells represent a valuable tool for treating cancers and infectious and inherited diseases; however, they are mainly short-lived in vivo. T-cell therapies would strongly benefit from gene transfer into long-lived persisting naive T cells or T-cell progenitors. Here we demonstrate that baboon envelope glycoprotein pseudotyped lentiviral vectors (BaEV-LVs) far outperformed other LV pseudotypes for transduction of naive adult and fetal interleukin-7-stimulated T cells. Remarkably, BaEV-LVs efficiently transduced thymocytes and T-cell progenitors generated by culture of CD34+ cells on Delta-like ligand 4 (Dll4). Upon NOD/SCIDγC-/- engraftment, high transduction levels (80%-90%) were maintained in all T-cell subpopulations. Moreover, T-cell lineage reconstitution was accelerated in NOD/SCIDγC-/- recipients after T-cell progenitor injection compared with hematopoietic stem cell transplantation. Furthermore, γC-encoding BaEV-LVs very efficiently transduced Dll4-generated T-cell precursors from a patient with X-linked severe combined immunodeficiency (SCID-X1), which fully rescued T-cell development in vitro. These results indicate that BaEV-LVs are valuable tools for the genetic modification of naive T cells, which are important targets for gene therapy. Moreover, they allowed for the generation of gene-corrected T-cell progenitors that rescued SCID-X1 T-cell development in vitro. Ultimately, the coinjection of LV-corrected T-cell progenitors and hematopoietic stem cells might accelerate T-cell reconstitution in immunodeficient patients.

Hypoxic niche-mediated regeneration of hematopoiesis in the engraftment window is dominantly affected by oxygen tension in the milieu.

The bone marrow (BM) microenvironment or the hematopoietic stem cell (HSC) niche is normally hypoxic, which maintains HSC quiescence. Paradoxically, transplanted HSCs rapidly proliferate in this niche. Pretransplant myelosuppression results in a substantial rise in oxygen levels in the marrow microenvironment due to reduced cellularity and consequent low oxygen consumption. Therefore, it may be construed that the rapid proliferation of the engrafted HSCs in the BM niche is facilitated by the transiently elevated oxygen tension in this milieu during the "engraftment window." To determine whether oxygen tension dominantly affects the regeneration of hematopoiesis in the BM niche, we created an "oxygen-independent hypoxic niche" by treating BM-derived mesenchymal stromal cells (BMSCs) with a hypoxia-mimetic compound, cobalt chloride (CoCl2) and cocultured them with BM-derived HSC-enriched cells under normoxic conditions (HSCs; CoCl2-cocultures). Cocultures with untreated BMSCs incubated under normoxia (control- cocultures) or hypoxia (1% O2; hypoxic-cocultures) were used as comparators. Biochemical analyses showed that though, both CoCl2 and hypoxia evoked comparable signals in the BMSCs, the regeneration of hematopoiesis in their respective cocultures was radically different. The CoCl2-BMSCs supported robust hematopoiesis, while the hypoxic-BMSCs exerted strong inhibition. The hematopoiesis-supportive ability of CoCl2-BMSCs was abrogated if the CoCl2-cocultures were incubated under hypoxia, demonstrating that the prevalent oxygen tension in the milieu dominantly affects the outcome of the HSC-BM niche interactions. Our data suggest that pharmacologically delaying the reestablishment of hypoxia in the BM may boost post-transplant regeneration of hematopoiesis.