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The spatio-temporal complexity of T cell differentiation from hematopoietic stem and progenitor cells (HSPCs) in the thymus has been challenging to recapitulate in vitro. While the OP9-DL1 monolayer co-culture system revolutionized thymopoiesis studies, serum-dependent monolayer systems can have inconsistent results. Our recently developed serum-free, 3D Artificial Thymic Organoid (ATO) system supports full in vitro T cell differentiation from human HSPCs and pluripotent stem cells; however, results using identical conditions with murine cells are suboptimal. Here, we demonstrate that culturing murine bone marrow (BM) HSPCs in a modified murine ATO system (M-ATO) consistently reproduces normal murine thymopoiesis. The M-ATO produces all T cell stages: from early thymic progenitors to mature T cell subsets, including TCRγδ+, single positive (SP8 and SP4) TCRαβ+, and FoxP3+ cells. RNA sequencing aligned M-ATO-derived populations with phenotypically identical primary thymocytes. M-ATO-derived CD3+TCRαβ+ SP cells are mature, functional T cells that exhibit a broad TCR Vβ repertoire and respond to TCR activation. M-ATOs initiated with Rag1-/- marrow produced the same differentiation block as seen in the endogenous thymus, and the transgenic Notch reporter (TNR) mouse model revealed that M-ATOs induce Notch signaling patterns in developing thymocytes similar to that in the thymus. Thymopoiesis can be established in M-ATOs from a range of BM and or thymic populations, including Lineage negative c-Kit+Sca-1+ (LSK), SLAM+ hematopoietic stem cells (HSC), lymphoid-primed multipotential progenitors (LMPP), common lymphoid progenitors (CLP) or early thymic progenitors (ETPs). The M-ATO is highly efficient at producing millions of T cells with a broad repertoire even when starting from purified single HSC or LSK cells. Thus, the M-ATO is a reproducible and efficient platform to study clonal murine T cell development and maturation from single HSCs and progenitors.

Neurocognitive function of pediatric patients is of great concern after hematopoietic stem cell transplantation (HSCT). We evaluated the neurocognitive function of pediatric patients pre-HSCT, 1, 3, and 5 years post-HSCT. All patients had a hematologic malignancy and received therapy to their central nervous system. Healthy siblings were tested as a comparison group. Pediatric patients with a hematologic malignancy did not have a significant decrease in their cognitive function before HSCT compared with their siblings except in areas of academic achievement. Our study population had significant declines in visual motor skills and memory test scores within the first year post-HSCT. By 3 years post-HSCT, there was an improvement in the visual motor development scores and memory scores, but there were new deficits in verbal skills. By 5 years post-HSCT, there were progressive declines in verbal skills (P=0.005), performance skills (0.04), and new deficits seen in long-term verbal memory scores (0.04). On the basis of the raw scores, most of these tests showed that patients had an inability to acquire new skills at a rate comparable to their age-matched healthy peers. However, long-term memory scores showed definite declines. The greatest decline in neurocognitive function occurred in those patients who received cranial irradiation either as part of their initial therapy or as part of their HSCT conditioning. Pediatric patients who received HSCT for hematologic malignancies have neurocognitive deficiencies that are both acute and chronic. Although some patients have acute deficits that appear and improve over time, other patients have progressive declines in neurocognitive function that are chronic.

Summary Advances in pluripotent stem cell and reprogramming technologies have given us the hope of generating hematopoietic stem cells (HSCs) in culture. To succeed, greater understanding of the self-renewing HSC during human development is required. We discovered that the glycophosphatidylinositol-anchored surface protein GPI-80 defines a subpopulation of human fetal liver hematopoietic stem/progenitor cells (HSPCs) with self-renewal ability. CD34 + CD38 lo/− CD90 + GPI-80 + HSPCs were the sole population that maintained proliferative potential and an undifferentiated state in stroma coculture and engrafted in immunodeficient mice. GPI-80 expression also enabled tracking of HSPCs once they emerged from endothelium and migrated between human fetal hematopoietic niches. GPI-80 colocalized on the surface of HSPCs with Integrin alpha-M (ITGAM), which in leukocytes cooperates with GPI-80 to support migration. Knockdown of GPI-80 or ITGAM was sufficient to compromise HSPC expansion in culture and engraftment in vivo. These findings indicate that human fetal HSCs employ mechanisms used in leukocyte adhesion and migration to mediate HSC self-renewal.

Metabolic diseases that have been treated by bone marrow transplantation (BMT) can be divided into three groups: (1) group I, those diseases in which the clinical symptomatology is restricted to cells of the lymphohematopoietic system, (2) group II, those diseases in which nonlymphohematopoietic cells are clinically affected but the central nervous system (CNS) is not affected, and (3) group III, those diseases in which the CNS is affected (Table 1). Group I diseases are all correctable by BMT, group II diseases may be correctable depending upon the biology of the primary disease, and group III diseases are still the center of controversy.

Patients with Gaucher disease suffer from a lack of functional glucocerebrosidase enzyme (Gc). Disease symptoms are a result of macrophage engorgement secondary to this enzyme deficiency. This study is designed to determine if cDNA encoding normal Gc can be introduced into macrophage precursors using a retroviral vector. CD34+ cells obtained from G-CSF mobilized peripheral blood stem cells or from bone marrow will be transduced ex vivo using one of the following three methods of transduction: 1) GlGc retroviral supernatant in the presence of autologous stroma over a period of 72 hours, 2) GlGc retroviral supernatant in the presence of interleukin-3, interleukin-6, stem cell factor and autologous stroma over a 72 hour period, 3) G1Gc retroviral supernatant in the presence of interleukin-3, interleukin-6, and stem cell factor over a 72 hour period. These transduced cells will be reinfused into the patient and the patient monitored for toxicities as well as evidence of successful gene transfer and expression. A total of twenty-four patients will be enrolled on the protocol. Patients will be assigned in equal numbers to each of six groups. The two sites participating are the National Institutes of Health and Childrens Hospital of Los Angeles.

The cytokine thrombopoietin (Tpo) plays a critical role in hematopoiesis by binding to the extracellular domain and inducing homodimerization of the intracellular signaling domain of its receptor, c-Mpl. Mpl homodimerization can also be accomplished by binding of a synthetic ligand to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. Unexpectedly, in contrast to Tpo stimulation, robust erythropoiesis is induced after dimerization of F36VMpl in human CD34+ progenitor cells. The goal of this study was to define the hematopoietic progenitor stages at which dimerization of intracellular Mpl induces erythropoiesis and the downstream molecular events that mediate this unanticipated effect. Dimerization (in the absence of erythropoietin and other cytokines) in human common myeloid progenitors and megakaryocytic erythroid progenitors caused a significant increase in CD34+ cells (p < .01) and induced all stages of erythropoiesis including production of enucleated red blood cells. In contrast, erythropoiesis was not seen with Tpo stimulation. CD34+ cell expansion was the result of increased cell cycling and survival (p < .05). Microarray profiling of CD34+ cells demonstrated that a unique transcriptional pattern is activated in progenitors by F36VMpl dimerization. Ligand-inducible dimerization of intracellular Mpl in human myeloerythroid progenitors induces progenitor expansion and erythropoiesis through molecular mechanisms that are not shared by Tpo stimulation of endogenous Mpl. Disclosure of potential conflicts of interest is found at the end of this article.

During steady-state hematopoiesis, the monocytic lineage, which includes macrophages, osteoclasts, monocytes, and monocyte-derived dendritic cells (moDCs), arises from a progenitor hierarchy in bone marrow. In humans, multipotent myeloid progenitors such as the granulocyte-monocyte progenitor (GMP) are proximal in this hierarchy, however the distal stages of monocytopoieis are unclear. We report here identification of a human clonogenic, monocytic-committed progenitor with a lineage negative, CD34+ CD38+ CD45RA+ CD123int CD115+ phenotype, which was developmentally conserved across bone marrow, cord blood, and fetal liver. Ex vivo analysis demonstrated a high cloning efficiency, and monocytic but not granulocytic CFU potential. Differentiation in the presence of M-CSF resulted in exclusive production of functional M2-like macrophages, whereas cultures modified with RANKL and GM-CSF/IL-4 resulted in highly enriched cultures of functional osteoclasts and moDCs, respectively. Transplantation into immunodeficient mice reconstituted bone marrow, splenic, pulmonary, and hepatic macrophage subsets, as well as circulating CD14+ monocytic cells. Of note, GMP depleted of CD115+ cells could regenerate the monocytic progenitor phenotype in vitro and in vivo, consistent with a step-wise program of lineage restriction during monocytopoiesis. Identification of the steady-state human monocytic progenitor offers opportunities for therapeutic expansion or modification of the monocytic lineage.

Due to a heightened awareness of the potential for adverse events during human gene therapy trials, it is important to document the safety of the vectors and to examine the potential for malignancy arising from insertional mutagenesis from integrating vectors, using detailed in vivo analyses. In the current studies, we assessed the potential for generation of replication-competent retrovirus (RCR) and insertional mutagenesis causing adverse events, in human cells transplanted into immune deficient mice. Both retroviral and lentiviral vectors were assessed, in a total of 630 recipient mice. Human hematopoietic and mesenchymal stem cells carrying two different Moloney-based vectors were co-transplanted into beige/nude/XID and NOD/SCID mice that are unable to reject cells that become transformed. A total of 481 mice transplanted with human cells transduced by Moloney-based vectors, and an additional 149 mice that had received human cells transduced by HIV-based lentiviral vectors were monitored daily for adverse events for 2–18 months post-transplantation. An “adverse event” was logged in the experiment, the mouse was immediately sacrificed, and an autopsy was performed if ANY signs of ill health were observed, including any of the following: weight loss, hunching, lethargy, rapid breathing, skin discoloration or irregularities, bloating, hemi-paresis, enlarged lymph nodes or visible solid tumors under the skin. The presence of tumors and organ or lymph node abnormalities, or progression to monoclonal expansion in hematopoietic cells were specifically sought. The organs, marrow, blood, and serum were banked. Tissue sections were prepared from all organs (and visible tumor, if present). DNA was isolated from marrow, organs, blood, and any tumors to determine whether vector elements were present. Of the 630 recipient mice, 117 had some evidence of adverse effects ranging from development of leukemia to scarred skin or discolored organs detected upon autopsy. Human leukemic cells were found infiltrating the murine liver, spleen, blood and lymph nodes in 4 of these 117 mice. No vector DNA was present. 37 of the mice had developed solid tumors, determined to be of murine origin with no vector DNA present or RCR detected in their serum or tumor tissue. Despite the injection of 459 million transduced cells throughout the experiments, the engineered human MSC never gave rise to solid tumors. None of the serum samples or affected tissues from these mice had vector integrants or produced RCR in the marker rescue assay. In summary, 4 of the 117 total adverse events were due to the spontaneous development of human leukemia and the remainder of the events were due to development of cancerous states in the murine tissues, with no evidence of retroviral vector involvement. There was no evidence of insertional mutagenesis causing human leukemia or solid tumors in any mouse. No RCR were detected in 117 serum samples analyzed by vector rescue assay. No adverse events were caused by the vectors and no mouse tested had HIV p24 capsid protein in their serum. The current studies provide an in vivo system to assess potential risk from RCR and from insertional mutagenesis when retroviral or lentiviral vectors are considered as candidates for human gene therapy.

In certain applications of gene therapy, regulated (rather than constitutive) gene expression will be crucial for proper function or optimal effect of the delivered gene. Therefore, an important goal of gene therapy is to be able to deliver genes so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Additionally, regulating transgene expression will be an important component of efforts for reducing risks of adverse advents associated with gene therapy with integrating vectors.

There are multiple applications for expansion of Hematopoietic Stem Cells (HSC) and progenitors in transplantation. Gene therapy for most hematopoietic diseases requires selective expansion of genetically corrected HSC to achieve therapeutic effects. Obtaining sufficient number of HSC (CD34+) is a limitation for use of cord blood (CB) in transplantation. Delayed immune recovery following HSC transplantation is associated with increased morbidity and mortality. Murine lymphoid recovery can be hastened by co-transplantation of Common Lymphoid Progenitors (CLP) with HSC, however, the rarity of human CLP in harvested products preclude their clinical use. We hypothesized that human HSC and CLP can be selectively and reversibly expanded by expression of a fusion protein comprised of the intracellular signaling domain of Thrombopoietin receptor (mpl), linked to a specific binding domain (F36V) for the chemical inducer of dimerization AP20187 (CID) (ARIAD Pharmaceuticals). Upon binding of CID to F36V, mpl signaling occurs. A lentiviral vector expressing the fusion protein (F36V-mpl) and a marker gene (GFP) was constructed and efficiently transduced and expressed in human CB HSC (CD34+CD38-CD7-) and CLP (CD34+CD38-CD7+). CID-induced mpl signaling in transduced human CLP maintained robust generation of total, B and NK cells for > 60 days, in cytokine free lymphoid cultures (N=4). Under these conditions transduced HSC cultures (N=4) maintained robust generation of total, B, NK cells for >120 day. Transduced HSC continued to generate clonogenic myelo-erythroid progenitors for > 120 days in ELTC-IC assay (N=4). These cytokine free in vitro assays indicate that CID-induced mpl signaling in human HSC and CLP induced prolonged survival and proliferation of transduced progenitors. Most of the cells generated in the presence of CID expressed GFP (mean 86% GFP+), indicating selective proliferation of transduced cells. Rapid decline in the number of cells expressing GFP was noticed upon withdrawal of CID, indicating reversible activation of mpl signaling in the transduced cells. In contrast, transduced human HSC and CLP cultured without CID, proliferated poorly and differentiated rapidly; viable cells were lost by day 22 of culture. To study whether cells stimulated by CID remained immunophenotypically and functionally primitive after dividing, cell divisions were tracked by labeling with the membrane dye PKH26. Proliferation index of transduced HSC was consistently higher in the presence of CID than in its absence. Human CD34+ cells that had undergone 3 divisions in the presence of CID and in absence of any cytokines, maintained a primitive CD34+ immunophenotype in vitro. In contrast, cells that divided in the absence of CID lost CD34 expression. Cells cultured ± CID were isolated after 3 divisions (based on PKH26 staining) and transplanted in equal numbers (40,000 cell/mouse) into sublethally ablated NOD/SCIDb2m −/− mice to assess function. Only CID-expanded cells were able to engraft, producing B lymphoid and myeloid progeny. Bone marrow harvested from the engrafted animals (N=3) contained clonogenic human myelo-erythroid progenitors (confirmed by Alu PCR of human specific CFU); cells cultured without CID failed to engraft. These studies show that CID-induced mpl signaling expands functionally primitive multipotent, engrafting human progenitors. This is a potential approach to selectively and reversibly expand transduced primitive human progenitors for use in cell therapy.