Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies.
We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34 ؉ cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m 2 ). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency. (Blood. 2012;120(18):3635-3646)
With their unique ability to differentiate into all cell types, embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation, we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100 -300 m) are the most proliferative, hold the greatest differentiation potential, and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation, we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates, we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system, similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation.hydrophobicity ͉ self-assembled monolayers ͉ serum-free differentiation T he potential of embryonic stem (ES) cells to differentiate into all specialized cell types has made them attractive models for studying the mechanisms of lineage commitment and has opened pathways for regenerative medicine (1). Various in vitro strategies have been developed for differentiation of ES cells into populations of specific cell types (2). Of these strategies, the formation of three-dimensional cell aggregates known as embryoid bodies (EBs) is a common and critical intermediate to the induction of lineage-specific differentiation (3, 4). In addition, lineage differentiation programs within the EB closely resemble lineage commitment in vivo in the developing embryo, further highlighting the importance of the ES cell-EB culture system (5-8).Although EBs can be generated through several methodologies, the suspension culture technique allows for easy access to the cultured EBs and can be scaled for expansion (9). In this method, EBs are formed when ES cells are removed from feeder contact and dispersed on low-attachment tissue culture plates, supplemented by culture medium absent of key factors necessary for the maintenance of undifferentiated ES cell growth. Lowattachment tissue culture plates typically use neutral, hydrophilic hydrogels to prevent protein adsorption and subsequent cell attachment, facilitating the initial aggregation of ES cells that is critical to EB formation (10). The cellular aggregates formed by this procedure will develop simple EBs that consist of an outer layer of endoderm cells within 2-4 days (3). At this point, two differentiation strategies can be applied. If suspension culture is continued, simple EBs will differentiate further to form cystic EBs that typically contain an inner layer of columnar ectodermlike cells and that accumulate fluid in the interior of the struct...
We have successfully designed and fabricated an integrated microfluidic platform, the hESC-microChip, which is capable of reproducible and quantitative culture and analysis of individual hESC colonies in a semi-automated fashion. In this device, a serpentine microchannel allows pre-screening of dissociated hESC clusters, and six individually addressable cell culture chambers enable parallel hESC culture, as well as multiparameter analyses in sequence. In order to quantitatively monitor hESC proliferation and pluripotency status in real time, knock-in hESC lines with EGFP driven by the endogenous OCT4 promoter were constructed. On-chip immunoassays of several pluripotency markers were carried out to confirm that the hESC colonies maintained their pluripotency. For the first time, our studies demonstrated well characterized hESC culture and analysis in a microfluidic setting, as well as a proof-of-concept demonstration of parallel/multiparameter/real-time/automated examination of self-renewal and differentiation in the same device.
Summary: Hematopoietic stem cells (HSCs) provide an attractive target for immunotherapy of cancer and leukemia by the introduction of genes encoding T-cell receptors (TCR) or chimeric antigen receptors (CARs) directed against tumor-associated antigens. HSCs engraft for long-term blood cell production and could provide a sustained source of targeted anti-cancer effector cells to sustain remissions. T cells produced de novo from HSCs may continuously replenish anti-tumor T cells that have become anergic or exhausted from ex vivo expansion or exposure to the intratumoral microenvironment. Additionally, transgenic T cells produced in vivo undergo allelic exclusion, preventing co-expression of an endogenous TCR that could mis-pair with the introduced TCR chains and blunt activity or even cause off-target reactivity. CAR-engineered HSCs may produce myeloid and NK cells in addition to T cells expressing the CAR, providing broader anti-tumor activity that arises quickly after transplant and does not solely require de novo thymopoiesis. Use of TCR- or CAR-engineered HSC would likely require cytoreductive conditioning to achieve long-term engraftment, and this approach may be used in clinical settings where autologous HSC transplant is being performed to add a graft-versus-tumor effect. Results of experimental and pre-clinical studies performed to date are reviewed.
Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34(+) HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8(+) and CD4(+) T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4-5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8(+) and CD4(+) T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34(+) HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.
IntroductionThe hematopoietic system is hierarchically organized, with a rare population of hematopoietic stem cells giving rise to specific progenitor cells and ultimately to diverse mature blood cell types. Lineage-specific fate decisions are intricately controlled by sets of master transcription factors. 1 For example, erythroid versus myeloid lineage differentiation is based on transcriptional crossantagonism between GATA-1 and PU.1. 2 PU.1 and C/EBP␣ are required for the generation of both macrophages and neutrophils. 3 In addition to transcription factors that are pivotal for hematopoietic cell lineage specification, several developmentally conserved signaling pathways, including Notch, Wnt, Sonic hedgehog, and TGF-, have also been implicated in regulating lineage determination. 4 MicroRNAs (miRs) are a recently discovered class of small (20-22 nt) noncoding RNAs that control gene expression posttranscriptionally by regulating mRNA translation or stability. 5 The roles of miRs in regulating the hematopoietic system are still poorly understood, although a series of recent studies have provided some perspective on this important issue. 6 There are several miRs, including miR-142, miR-181a, and miR-223, that are prominently expressed in hematopoietic cells. 7,8 Ectopic expression of miR-181a in hematopoietic stem/progenitor cells promotes B-cell differentiation, whereas overexpression of miR-142 and miR-223 leads to an increase in the proportion of T-lineage cells. 7 Some studies have not only delineated a role for miRs in hematopoiesis but also uncovered their molecular mechanisms of action involving regulation of lineage determining transcription factors. For example, the combination of gain-of-function and loss-of-function strategies has defined the requirement of miR-150 in B-cell differentiation by targeting the transcription factor c-Myb. 9 During monocytopoiesis, down-regulation of miRs 17-5p-20a-106a relieves the suppression of AML1, thus facilitating monocytic differentiation and maturation. 10 In addition to the role of specific miRs, the general requirement of miRs in hematopoiesis has been demonstrated, as deletion of Dicer in the thymus results in aberrant T-cell development in the murine system. 11,12 There is expanding literature suggesting that miRs also play an important role in the pathology of hematologic malignancies as tumor suppressor genes or oncogenes. For example, both miR-15a and miR-16 are deleted or down-regulated in the majority of chronic lymphocytic leukemia cases, 13 whereas C57BL6 mice transplanted with murine hematopoietic progenitor cells overexpressing miR-155 developed a myeloproliferative disorder. 14 Given compelling evidence that miRs are essential in the hematopoietic system, we sought to elucidate the potential roles of miRs in early human hematopoietic development. Insights into hematopoietic development have largely relied on findings from gene knockout and transgenic mouse models as well as in other model organisms. Because of the inaccessibility of human embryos,...
Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [ 18 F]-L-FMAU (1-(2-deoxy-2- 18 fluoro-β-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.