Purified enzymes improve isolation and characterization of the adult thymic epithelium

Seach, Natalie Louise; Wong, Kahlia; Hammett, Maree Vanessa; Boyd, Richard; Chidgey, Ann Patricia

doi:10.1016/j.jim.2012.07.023

Cited by 67 publications

(63 citation statements)

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“…Other groups have also reported their new methods for thymic stromal cell isolation by modifying Gray's method 30,36 , although cell yield was not significantly increased. Recently, Chidgey and colleagues independently reported a TEC protocol using Liberase digestion and they also obtained effective release of TECs 37 . However, the first thymocyte release step of their protocol has the potential to discard about 1.6 x 10 4 TECs from each thymus 29 , most of which are cTECs.…”

Section: Discussionmentioning

confidence: 99%

“…However, the first thymocyte release step of their protocol has the potential to discard about 1.6 x 10 4 TECs from each thymus 29 , most of which are cTECs. Due to density gradient enrichment causing loss of smaller stromal cells, an AutoMACSbased CD45-microbead depletion is popularly used for enrichment of TECs prior to FACS purification 29,36,37 . Compared to the AutoMACSbased enrichment using CD45-microbead depletion protocols 29 , the panning enrichment obtained higher recovery rates of TECs.…”

Section: Discussionmentioning

confidence: 99%

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Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Xing

Hogquist

2014

JoVE

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The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cellcell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Xing

Hogquist

2014

JoVE

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“…The FOXN1-TAT gene was amplified using the sense primer above and antisense primer 5 -CTCGAGTCAGCGGCGGCGCT GGCGGCGTTTCTTGCGGCCATAAGCCAGAGCCAATGGCTTTG-3 encoding YGRKKRRQRRR from the HIV TAT [47][48][49][50][51][52][53][54][55][56][57] . The entire DNA fragment for FOXN1-TAT was cloned into the expression vector pET-45b(+) (Novagen, Gibbstown, NJ, USA) using the In-Fusion Advantage PCR Cloning Kit (Clontech, Mountain View, CA, USA) with two primers (5 -CCATCACGTGGGTACCGTGTCGC TACTCCC-3 and 5 -CTTTACCAGACTCGAGTCAGCGGCGGCGCT-3 ) according to the manufacturer's instructions.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Nutley NJ, USA) and 0.02% (w/v) DNAse I (Roche) with regular and gentle agitation as described [56]. CD45 − cells were negatively selected by CD45 microbeads, and EpCAM + cells were then positively selected by EpCAM microbeads (Miltenyi Biotec, San Diego, CA, USA).…”

Section: Tec Isolationmentioning

confidence: 99%

“…The use of rFOXN1 protein may overcome these adversities. Because the cell-penetrating peptides or protein transduction domain (PTD) embedded in the HIV transactivator of transcription (TAT) protein (amino acids [47][48][49][50][51][52][53][54][55][56][57] has been shown to successfully mediate the induction of protein into mammalian cells in vitro and in vivo [28,29], we cloned and expressed rFOXN1 protein fused with TAT PTD. We demonstrate here that the rFOXN1 can enter the cytoplasm and nucleus.…”

Section: Introductionmentioning

confidence: 99%

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FOXN1 recombinant protein enhances T‐cell regeneration after hematopoietic stem cell transplantation in mice

Song

Zhu

et al. 2016

Eur J Immunol

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A prolonged period of T-cell recovery is the major challenge in hematopoietic stem cell transplantation (HSCT). Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T-cell generation. However, TECs undergo degeneration over time. FOXN1 plays a critical role in TEC development and is required to maintain adult TECs for thymopoiesis. To investigate the potential application of FOXN1, we have cloned and expressed recombinant FOXN1 protein (rFOXN1) that was fused with cellpenetrating peptides. We show here that the rFOXN1 protein can translocate from the cell surface into the cytoplasm and nucleus. Administration of rFOXN1 into both congenic and allogeneic HSCT recipient mice increased the number of TECs, resulting in enhanced thymopoiesis that led to an increased number of functional T cells in the periphery. The increased number of TECs is due to the enhanced survival and proliferation of TECs. Our results suggest that rFOXN1 has the potential to be used in enhancing T-cell regeneration in patients following HSCT.Keywords: FOXN1 r Hematopoietic stem cell transplantation r T-cell generation r Thymic epithelial cells r Thymus Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionHSCT is widely used in the treatment of hematopoietic and nonhematopoietic diseases. However, this procedure often suffers from a long period of T-cell recovery, which contributes to increased incidences of infection and relapses of cancer [1][2][3][4][5][6]. Therefore, strategies to enhance T-cell regeneration after HSCT would be greatly beneficial.Correspondence: Dr. Laijun Lai e-mail: laijun.lai@uconn.edu T-cell development occurs primarily in the thymus, and is critically dependent on the thymic microenvironment, in which TECs are the major component. The importance of TECs in thymocyte development has been highlighted by the fact that abnormal TEC development results in immunodeficiency and autoimmunity [7,8]. It is known that TECs undergo a qualitative and quantitative loss over time [8][9][10]. In addition, radiation, chemotherapy, immunosuppressive drugs, and infections, etc. may injure the TECs. Therefore, strategies to restore TECs should lead to enhanced T-cell regeneration and adaptive immunity.FOXN1 is a member of the winged helix/forkhead box transcription factor family. It is generally acknowledged that FOXN1 is a pivotal regulator for TEC development [11][12][13][14][15][16][17][18][19]. Mice homozygous for loss-of-function mutation in FOXN1 display the 'nude' phenotype (FOXN1 nu/nu ), which is characterized by congenital athymia and hairlessness. A mutation in the human FOXN1 gene also results in a nude phenotype [20,21]. FOXN1 is expressed in fetal thymus and postnatal TECs and its expression is progressively down-regulated with aging [15,16,[22][23][24][25]. FOXN1 is required not only for TEC development in fetal thymus, but also for maintenance of the postnatal thymus [15,16,23,24,26]. The reduced expression or indu...

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Isolation of Thymic Epithelial Cells and Analysis by Flow Cytometry

Jain

Gray

2014

CP in Immunology

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The epithelial cells of the thymus govern the differentiation of hematopoietic precursors into T cells, which are critical for acquired immunity. Thymic epithelial cells (TEC) provide molecular cues that direct precursor recruitment, commitment to the T cell lineage, thymocyte proliferation, and the processes of positive and negative selection. Despite the importance of TEC to the immune system, fundamental questions regarding their differentiation, turnover, and function throughout life remain unanswered. This knowledge gap is largely due to technical difficulties in isolating, quantifying, and purifying this rare cell type. Here, we describe methods for the enzymatic digestion of the thymus to obtain single-cell suspensions of TEC, their analysis by flow cytometry, enrichment using magnetic beads, and purification for a variety of downstream applications.

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Purified enzymes improve isolation and characterization of the adult thymic epithelium

Cited by 67 publications

References 47 publications

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

FOXN1 recombinant protein enhances T‐cell regeneration after hematopoietic stem cell transplantation in mice

Isolation of Thymic Epithelial Cells and Analysis by Flow Cytometry

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