The molecular and cellular signals that guide T-cell development from hematopoietic stem and progenitor cells (HSPCs) remain poorly understood. The thymic microenvironment integrates multiple niche molecules to potentiate T-cell development in vivo. Recapitulating these signals in vitro in a stromal cell-free system has been challenging and limits T-cell generation technologies. Here, we describe a fully defined engineered in vitro niche capable of guiding T-lineage development from HSPCs. Synergistic interactions between Notch ligand Delta-like 4 and vascular cell adhesion molecule 1 (VCAM-1) were leveraged to enhance Notch signaling and progenitor T-cell differentiation rates. The engineered thymus-like niche enables in vitro production of mouse Sca-1cKit and human CD34 HSPC-derived CD7 progenitor T-cells capable of in vivo thymus colonization and maturation into cytokine-producing CD3 T-cells. This engineered thymic-like niche provides a platform for in vitro analysis of human T-cell development as well as clinical-scale cell production for future development of immunotherapeutic applications.

Pluripotent stem cells (PSCs) can form any specialized cell type found in the body making them an excellent tool for regenerative medicine applications. Directed differentiation of PSCs into specific phenotypes can be accomplished by introducing specific chemical cues such as the small molecule retinoic acid (RA). Expressed in the developing nervous system, RA can induce differentiation of PSCs into neural phenotypes including neurons. In this study, we encapsulated all-trans RA within poly (ɛ-caprolactone) (PCL) microspheres to generate controlled morphogen release over 28 days. RA/PCL microspheres less than ~10 µm in diameter were readily incorporated within the interstitial sites of human induced pluripotent stem cell (hiPSC) aggregates. After 5 days of culture, the microspheres did not induce cytotoxic effects and the hiPSC aggregates containing microspheres showed a decrease in the pluripotency marker SSEA-4. After 7 days of culture on laminin surfaces, aggregates expressed the neuronal marker TUJ1 and displayed extended neurite outgrowth. This approach provides consistent RA delivery throughout the aggregate and could be an effective strategy for differentiating cells in vivo.Overall, our results demonstrate that it is possible to combine hiPSC aggregates with RA/PCL microspheres for neural tissue engineering applications.

T cells show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to provide a renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains an important challenge. Here, we report an efficient serum- and feeder-free system for differentiating human PSCs into hematopoietic progenitors and T cells. This fully defined approach allowed us to study the impact of individual proteins on blood emergence and differentiation. Providing DLL4 and VCAM1 during the endothelial-to-hematopoietic transition enhanced downstream progenitor T cell output by ~80-fold. These two proteins synergized to activate notch signaling in nascent hematopoietic stem and progenitor cells, and VCAM1 additionally promoted an inflammatory transcriptional program. We also established optimized medium formulations that enabled efficient and chemically defined maturation of functional CD8αβ + , CD4 − , CD3 + , TCRαβ + T cells with a diverse TCR repertoire.

No abstract

Ongoing clinical trials are evaluating the use of stem cells as a way to treat traumatic spinal cord injury (SCI). However, the inhibitory environment present in the injured spinal cord makes it challenging to achieve the survival of these cells along with desired differentiation into the appropriate phenotypes necessary to regain function. Transplanting stem cells along with an instructive biomaterial scaffold can increase cell survival and improve differentiation efficiency. This study reviews the literature discussing different types of instructive biomaterial scaffolds developed for transplanting stem cells into the injured spinal cord. We have chosen to focus specifically on biomaterial scaffolds that direct the differentiation of neural stem cells and pluripotent stem cells since they offer the most promise for producing the cell phenotypes that could restore function after SCI. In terms of biomaterial scaffolds, this article reviews the literature associated with using hydrogels made from natural biomaterials and electrospun scaffolds for differentiating stem cells into neural phenotypes. It then presents new data showing how these different types of scaffolds can be combined for neural tissue engineering applications and provides directions for future studies.

The generation of T-cells from stem cells in vitro could provide an alternative source of cells for immunotherapies. T-cell development from hematopoietic stem and progenitor cells (HSPCs) is tightly regulated through Notch pathway activation by Delta-like (DL) ligands 1 and 4. Other molecules, such as stem cell factor (SCF) and interleukin (IL)-7, play a supportive role in regulating the survival, differentiation, and proliferation of developing T-cells. Numerous other signaling molecules influence T-lineage development in vivo, but little work has been done to understand and optimize their use for T-cell production. Using a defined engineered thymic niche system, we undertook a multi-stage statistical learning-based optimization campaign and identified IL-3 and tumor necrosis factor α (TNFα) as a stage- and dose-specific enhancers of cell proliferation and T-lineage differentiation. We used this information to construct an efficient three-stage process for generating conventional TCRαβ+CD8+ T-cells expressing a diverse TCR repertoire from blood stem cells. Our work provides new insight into T-cell development and a robust system for generating T-cells to enable clinical therapies for treating cancer and immune disorders.

T cells are key mediators of the adaptive immune response and show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to serve as a consistent and renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains a significant challenge. Here, we developed an efficient serum- and feeder-free protocol for differentiating human PSCs into hematopoietic progenitors and T cells. This defined method allowed us to study the impact of individual recombinant proteins on blood emergence and lineage potential. We demonstrate that the presence of DLL4 and VCAM1 during the endothelial-to-hematopoietic transition (EHT) enhances downstream progenitor T cell output by >80-fold. Using single cell transcriptomics, we showed that these two proteins synergise to drive strong notch signalling in nascent hematopoietic stem and progenitor cells and that VCAM1 additionally drives a pro-inflammatory transcriptional program. Finally, we applied this differentiation method to study the impact of cytokine concentration dynamics on T cell maturation. We established optimised media formulations that enabled efficient and chemically defined differentiation of CD8αβ+, CD4-, CD3+, TCRαβ+ T cells from PSCs.

T-cell development from hematopoietic stem and progenitor cells (HSPCs) is tightly regulated through Notch pathway activation by the Notch ligands Delta-like (DL) 1 and 4 and Jagged-2. Other molecules, such as stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L) and interleukin (IL)-7, play a supportive role in regulating the survival, differentiation, and proliferation of developing progenitor (pro)T-cells. Numerous other signaling molecules are known to instruct T-lineage development in vivo, but little work has been done to optimize their use for T-cell production in vitro. Using a defined T-lineage differentiation assay consisting of plates coated with the Notch ligand DL4 and adhesion molecule VCAM-1, we performed a cytokine screen that identified IL-3 and tumor necrosis factor α (TNFα) as enhancers of proT-cell differentiation and expansion. Mechanistically, we found that TNFα induced T-lineage differentiation through the positive regulation of T-lineage genes GATA3, TCF7, and BCL11b. TNFα also synergized with IL-3 to induce proliferation by upregulating the expression of the IL-3 receptor on CD34+ HSPCs, yielding 753.2 (532.4-1026.9; 5-95 percentile)-fold expansion of total cells after 14 days compared to 8.9 (4.3-21.5)-fold expansion in conditions without IL-3 and TNFα. We then optimized cytokine concentrations for T-cell maturation. Focusing on T-cell maturation, we used quantitative models to optimize dynamically changing cytokine requirements and used these to construct a three-stage assay for generating CD3+CD4+CD8+ and CD3+CD4−CD8+ T-cells. Our work provides new insight into T-cell development and a robust in vitro assay for generating T-cells to enable clinical therapies for treating cancer and immune disorders.