Extrathymic T cell differentiation in vitro from human CD34+ stem cells

Abstract

Although it is well established that T cells are derived from CD34 ؉ stem cells in vivo, and that T cells can develop in the absence of a functioning thymus, it has not proven possible thus far to generate human T cells in vitro from CD34 ؉ cells in the absence of any thymic influence. We now present a limiting dilution cloning culture system that supports the differentiation of highly purified human CD34 ؉ cells to CD3 ؉ T cells in vitro in the complete absence of any thymic components. The culture system features the use of a serum-free medium supplemented with a cocktail of cytokines including flt-3 ligand, interleukin-3 (IL-3), stem cell factor (SCF), and IL-2. CD4 ؉ T cell clones capable of mitogen-stimulated proliferation and response to IL-2, and expressing a varied TCR-V␤ repertoire were obtained under these conditions. This culture system therefore supports human T lymphopoiesis in the absence of any thymic influence and may prove useful for the evaluation of extrathymic T cell differentiation in vitro. J. Leukoc. Biol. 64: 733-739; 1998.

Microsatellite Instability and Compromized Mismatch Repair Gene Expression During In Vitro Passaging of Monoclonal Human T Lymphocytes

“…Trizygosity, changes at all of the analyzed sequences and differences among clones obtained from the same donor were found very frequently, suggesting instability also related to the cell type in addition to in vitro proliferation and aging. This may suggest that the efficiency of the MMR system is already maximal in mature T cells, but that it is less efficient in CD34 ϩ progenitors due to their maturation stage and requirement for differentiation to T cells in vitro and not in the normal in vivo environment 26,28 ; indeed, early progenitors are highly proliferative and, during this period, most susceptible to DNA damage. 39 Maturation-dependent alterations in DNA repair function have been demonstrated for the lymphohematopoietic system in association with shifts in DNA re-pair gene expression profiles.…”

“…Genotyping was done by standard polymerase chain reaction (PCR) followed by analysis on an automated DNA sequencer. Representative chromatograms of PCR products are shown (upper panels: D2S123(2p16), a (CA) 24 repeat located in the MLH1 gene; lower panels: BAT26(2p16), a (A) 26 repeat located in intron 5 of the MSH2 gene). Allele lengths in base pairs are indicated.…”

“…[26][27][28] Microsatellite instability analysis Total DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN GmbH, Germany). CD4, VWA, and Fes loci were typed as described.…”

Nanostructured Bifunctional Hydrogels as Potential Instructing Platform for Hematopoietic Stem Cell Differentiation

“…Efforts to induce T cell differentiation in vitro have been limited mainly due to the fact that the thymus, the organ in which T cells develop, is a complex three-dimensional network of epithelial cells (Holländer et al, 2006). This thymic microenvironment determines T cell lineage commitment via signals critical for T cell generation mediated for instance by Notch ligands Delta-like1 (DLL1) and DLL4, interleukin 7 (IL-7), FMS-like tyrosine kinase 3 ligand (Flt3L), and stem cell factor (SCF) (Pawelec et al, 1998;Besseyrias et al, 2007;Hozumi et al, 2008). Among others, Notch signaling plays an essential role in T cell development.…”

Toward Development and Production of Human T Cells in Swine for Potential Use in Adoptive T Cell Immunotherapy

“…New T cells can also be generated in vitro. [18][19][20][21][22] For example, Poznansky et al 22 seeded CD34 þ or AC133 þ cells on CellFoam coated with thymic stroma. These three-dimensional constructs supported T cell development more efficiently than two-dimensional constructs (*60% of CD45 þ cells).…”