GNTI-122: an autologous antigen-specific engineered Treg cell therapy for type 1 diabetes.

Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving ß cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.

A chemically inducible IL-2 receptor signaling complex allows for effective in vitro and in vivo selection of engineered CD4+ T cells.

Engineered T cells represent an emerging therapeutic modality. However, complex engineering strategies can present a challenge for enriching and expanding therapeutic cells at clinical scale. In addition, lack of in vivo cytokine support can lead to poor engraftment of transferred T cells, including regulatory T cells (Treg). Here, we establish a cell-intrinsic selection system that leverages the dependency of primary T cells on IL-2 signaling. FRB-IL2RB and FKBP-IL2RG fusion proteins were identified permitting selective expansion of primary CD4+ T cells in rapamycin supplemented medium. This chemically inducible signaling complex (CISC) was subsequently incorporated into HDR donor templates designed to drive expression of the Treg master regulator FOXP3. Following editing of CD4+ T cells, CISC+ engineered Treg (CISC EngTreg) were selectively expanded using rapamycin and maintained Treg activity. Following transfer into immunodeficient mice treated with rapamycin, CISC EngTreg exhibited sustained engraftment in the absence of IL-2. Furthermore, in vivo CISC engagement increased the therapeutic activity of CISC EngTreg. Finally, an editing strategy targeting the TRAC locus permitted generation and selective enrichment of CISC+ functional CD19-CAR-T cells. Together, CISC provides a robust platform to achieve both in vitro enrichment and in vivo engraftment and activation, features likely beneficial across multiple gene-edited T cell applications.

Assessment of Endothelial-to-Hematopoietic Transition of Individual Hemogenic Endothelium and Bulk Populations in Defined Conditions.

Endothelial-to-hematopoietic transition (EHT) is a unique morphogenic event in which flat, adherent hemogenic endothelial (HE) cells acquire round, non-adherent blood cell morphology. Investigating the mechanisms of EHT is critical for understanding the development of hematopoietic stem cells (HSCs) and the entirety of the adult immune system, and advancing technologies for manufacturing blood cells from human pluripotent stem cells (hPSCs). Here we describe a protocol to (a) generate and isolate subsets of HE from hPSCs, (b) assess EHT and hematopoietic potential of HE subsets in bulk cultures and at the single-cell level, and (c) evaluate the role of NOTCH signaling during HE specification and EHT. The generation of HE from hPSCs and EHT bulk cultures are performed in xenogen- and feeder-free system, providing the unique advantage of being able to investigate the role of individual signaling factors during EHT and the definitive lympho-myeloid cell specification from hPSCs.

Ancestral Stem Cell Reprogramming Genes Active in Hemichordate Regeneration.

Hemichordate enteropneust worms regenerate extensively in a manner that resembles the regeneration for which planaria and hydra are well known. Although hemichordates are often classified as an extant phylogenetic group that may hold ancestral deuterostome body plans at the base of the deuterostome evolutionary line leading to chordates, mammals, and humans, extensive regeneration is not known in any of these more advanced groups. Here we investigated whether hemichordates deploy functional homologs of canonical Yamanaka stem cell reprogramming factors, Oct4, Sox2, Nanog, and Klf4, as they regenerate. These reprogramming factors are not expressed during regeneration of limbs, fins, eyes or other structures that represent the best examples of regeneration in chordates. We first examined Ptychodera flava EST libraries and identified Pf-Pou3, Pf-SoxB1, Pf-Msxlx, and Pf-Klf1/2/4 as most closely related to the Yamanaka factors, respectively. In situ hybridization analyses revealed that all these homologs are expressed in a distinct manner during head regeneration. Furthermore, Pf-Pou3 partially rescued the loss of endogenous Oct4 in mouse embryonic stem cells in maintaining the pluripotency gene expression program. Based on these results, we propose that hemichordates may have co-opted these reprogramming factors for their extensive regeneration or that chordates may have lost the ability to mobilize these factors in response to damage. The robustness of these pluripotency gene circuits in the inner cell mass and in formation of induced pluripotent stem cells from mammalian somatic cells shows that these programs are intact in humans and other mammals and that these circuits may respond to as yet unknown gene regulatory signals, mobilizing full regeneration in hemichordates.

SOX17 integrates HOXA and arterial programs in hemogenic endothelium to drive definitive lympho-myeloid hematopoiesis.

SOX17 has been implicated in arterial specification and the maintenance of hematopoietic stem cells (HSCs) in the murine embryo. However, knowledge about molecular pathways and stage-specific effects of SOX17 in humans remains limited. Here, using SOX17-knockout and SOX17-inducible human pluripotent stem cells (hPSCs), paired with molecular profiling studies, we reveal that SOX17 is a master regulator of HOXA and arterial programs in hemogenic endothelium (HE) and is required for the specification of HE with robust lympho-myeloid potential and DLL4+CXCR4+ phenotype resembling arterial HE at the sites of HSC emergence. Along with the activation of NOTCH signaling, SOX17 directly activates CDX2 expression, leading to the upregulation of the HOXA cluster genes. Since deficiencies in HOXA and NOTCH signaling contribute to the impaired in vivo engraftment of hPSC-derived hematopoietic cells, the identification of SOX17 as a key regulator linking arterial and HOXA programs in HE may help to program HSC fate from hPSCs.

Production, safety and efficacy of iPSC-derived mesenchymal stromal cells in acute steroid-resistant graft versus host disease: a phase I, multicenter, open-label, dose-escalation study.

The therapeutic potential of donor-derived mesenchymal stromal cells (MSCs) has been investigated in diverse diseases1, including steroid-resistant acute graft versus host disease (SR-aGvHD)2. However, conventional manufacturing approaches are hampered by challenges with scalability and interdonor variability, and clinical trials have shown inconsistent outcomes3,4. Induced pluripotent stem cells (iPSCs) have the potential to overcome these challenges, due to their capacity for multilineage differentiation and indefinite proliferation5,6. Nonetheless, human clinical trials of iPSC-derived cells have not previously been completed. CYP-001 (iPSC-derived MSCs) is produced using an optimized, good manufacturing practice (GMP)-compliant manufacturing process. We conducted a phase 1, open-label clinical trial (no. NCT02923375) in subjects with SR-aGvHD. Sixteen subjects were screened and sequentially assigned to cohort A or cohort B (n = 8 per group). One subject in cohort B withdrew before receiving CYP-001 and was excluded from analysis. All other subjects received intravenous infusions of CYP-001 on days 0 and 7, at a dose level of either 1 × 106 cells per kg body weight, to a maximum of 1 × 108 cells per infusion (cohort A), or 2 × 106 cells per kg body weight, to a maximum dose of 2 × 108 cells per infusion (cohort B). The primary objective was to assess the safety and tolerability of CYP-001, while the secondary objectives were to evaluate efficacy based on the proportion of participants who showed a complete response (CR), overall response (OR) and overall survival (OS) by days 28/100. CYP-001 was safe and well tolerated. No serious adverse events were assessed as related to CYP-001. OR, CR and OS rates by day 100 were 86.7, 53.3 and 86.7%, respectively. The therapeutic application of iPSC-derived MSCs may now be explored in diverse inflammatory and immune-mediated diseases.

Generation of T cells from Human and Nonhuman Primate Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This article outlines several protocols related to generation of T cells from human and non-human primate (NHP) PSCs, including initial hematopoietic differentiation of PSC on OP9 feeders or defined conditions, followed by coculture of the OP9-DLL4 cells with the PSC-derived hematopoietic progenitors (HPs), leading to efficient differentiation to T lymphocytes. In addition, we describe a protocol for robust T cell generation from hPSCs conditionally expressing ETS1. The presented protocols provide a platform for T cell production for disease modeling and evaluating their use for immunotherapy in large animal models.

Arterial identity of hemogenic endothelium: a key to unlock definitive hematopoietic commitment in human pluripotent stem cell cultures.

Human pluripotent stem cells (hPSCs) have been suggested as a potential source for the de novo production of blood cells for transfusion, immunotherapies, and transplantation. However, even with advanced hematopoietic differentiation methods, the primitive and myeloid-restricted waves of hematopoiesis dominate in hPSC differentiation cultures, whereas cell surface markers to distinguish these waves of hematopoiesis from lympho-myeloid hematopoiesis remain unknown. In the embryo, hematopoietic stem cells (HSCs) arise from hemogenic endothelium (HE) lining arteries, but not veins. This observation led to a long-standing hypothesis that arterial specification is an essential prerequisite to initiate the HSC program. It has also been established that lymphoid potential in the yolk sac and extraembryonic vasculature is mostly confined to arteries, whereas myeloid-restricted hematopoiesis is not specific to arterial vessels. Here, we review how the link between arterialization and the subsequent definitive multilineage hematopoietic program can be exploited to identify HE enriched in lymphoid progenitors and aid in in vitro approaches to enhance the production of lymphoid cells and potentially HSCs from hPSCs. We also discuss alternative models of hematopoietic specification at arterial sites and recent advances in our understanding of hematopoietic development and the production of engraftable hematopoietic cells from hPSCs.

NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells.

NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144+CD43-CD73-DLL4+Runx1 + 23-GFP+ arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs.

Activation of the Arterial Program Drives Development of Definitive Hemogenic Endothelium with Lymphoid Potential.

Understanding the pathways guiding the development of definitive hematopoiesis with lymphoid potential is essential for advancing human pluripotent stem cell (hPSC) technologies for the treatment of blood diseases and immunotherapies. In the embryo, lymphoid progenitors and hematopoietic stem cells (HSCs) arise from hemogenic endothelium (HE) lining arteries but not veins. Here, we show that activation of the arterial program through ETS1 overexpression or by modulating MAPK/ERK signaling pathways at the mesodermal stage of development dramatically enhanced the formation of arterial-type HE expressing DLL4 and CXCR4. Blood cells generated from arterial HE were more than 100-fold enriched in T cell precursor frequency and possessed the capacity to produce B lymphocytes and red blood cells expressing high levels of BCL11a and ß-globin. Together, these findings provide an innovative strategy to aid in the generation of definitive lymphomyeloid progenitors and lymphoid cells from hPSCs for immunotherapy through enhancing arterial programming of HE.

A human VE-cadherin-tdTomato and CD43-green fluorescent protein dual reporter cell line for study endothelial to hematopoietic transition.

Human embryonic stem cell line WA01 was genetically modified using zinc-finger nucleases and the PiggyBac/transponson system to introduce a fluorescence reporter for VE-cadherin (VEC; tdTomato) and CD43 (eGFP). Phenotypic and functional assays for pluripotency revealed the modified hES cell reporter lines remained normal. When the cells were differentiated into hematoendothelial lineages, either by directed differentiation or direct reprogramming, flow cytometric and fluorescence microscopy showed that VEC+ endothelial cells express tdTomato and CD43+ hematopoietic progenitors express eGFP.

Tenascin C promotes hematoendothelial development and T lymphoid commitment from human pluripotent stem cells in chemically defined conditions.

The recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs). However, the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells. Here, we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC), an extracellular matrix protein associated with HSC niches, strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders, including the formation of VE-Cadherin(+)CD73(-)CD235a/CD43(-) HE and hematopoietic progenitors with myeloid and T lymphoid potential.

Regeneration in the hemichordate Ptychodera flava.

When the body of P. flava is severed, the animal has the ability to regenerate its missing anterior or posterior as appropriate. We have focused on anterior regeneration when the head and branchial regions are severed from the body of the worm. After transection, the body wall contracts and heals closed in 2 to 3 days. By the third day a small blastema is evident at the point of closure. The blastema grows rapidly and begins the process of differentiating into a head with a proboscis and collar. At 5 days the blastema has increased greatly in size and differentiated into a central bulb, the forming proboscis, and two lateral crescents, the forming collar. Between 5 and 7 days a mouth opens ventral to the differentiating blastema. Over the next few days the lateral crescents extend to encircle the proboscis and mouth, making a fully formed collar. By 10 to 12 days a new head, sized to fit the worm's body, has grown attached to the severed site. At about this time the animal regains apparently normal burrowing behavior. After the head is formed, a second blastema-like area appears between the new head and the old body and a new branchial region is inserted by regeneration from this blastema over the next 2 to 3 weeks. The regenerating tissues are unpigmented and whitish such that in-situ hybridization can be used to study the expression of genes during the formation of new tissues.