Generation of mTert-GFP mice as a model to identify and study tissue progenitor cells

Abstract

Stem cells hold great promise for regenerative medicine, but remain elusive in many tissues in part because universal markers of "stemness" have not been identified. The ribonucleoprotein complex telomerase catalyzes the extension of chromosome ends, and its expression is associated with failure of cells to undergo cellular senescence. Because such resistance to senescence is a common characteristic of many stem cells, we hypothesized that telomerase expression may provide a selective biomarker for stem cells in multiple tissues. In fact, telomerase expression has been demonstrated within hematopoietic stem cells. We therefore generated mouse telomerase reverse transcriptase (mTert)-GFP-transgenic mice and assayed the ability of mTert-driven GFP to mark tissue stem cells in testis, bone marrow (BM), and intestine. mTert-GFP mice were generated by using a two-step embryonic stem cell-based strategy, which enabled primary and secondary screening of stably transfected clones before blastocyst injection, greatly increasing the probability of obtaining mTert reporter mice with physiologically appropriate regulation of GFP expression. Analysis of adult mice showed that GFP is expressed in differentiating male germ cells, is enriched among BM-derived hematopoietic stem cells, and specifically marks long-term BrdU-retaining intestinal crypt cells. In addition, telomerase-expressing GFP(+) BM cells showed long-term, serial, multilineage BM reconstitution, fulfilling the functional definition of hematopoietic stem cells. Together, these data provide direct evidence that mTert-GFP expression marks progenitor cells in blood and small intestine, validating these mice as a useful tool for the prospective identification, isolation, and functional characterization of progenitor/stem cells from multiple tissues.

Fig. 1.

mTert-GFP expression during EB formation. (a) Representative undifferentiated ES cell colony stably transfected with mTert-GFP. (b) Representative EB 7 days after LIF withdrawal. Note the outer layer of differentiated cells no longer expressing GFP. (c) Representative EB after 48 h of DMSO exposure and 7 days of LIF withdrawal. Note lack of GFP in the differentiated cell outgrowth. Each panel is a merge of Nomarski imaging (Upper Left Insets), DAPI stained nuclei (Upper Center Insets), and GFP expression (Upper Right Insets). (Magnification: ×20.)

Fig. 2.

mTert-GFP expression in testis. (a) Western blotting was performed by using whole testis protein extract from WT, mTert-GFP, or Actin-GFP mice and rabbit anti-GFP antibody. The blot was stripped and reblotted by using anti-Actin antibody as a loading control. (b) Representative FACS analysis for GFP expression performed on single-cell isolates from seminiferous tubules of adult mTert-GFP and WT testes. Cells were gated into 1N, 2N, and 4N populations. The dashed line illustrates the GFP threshold as defined by WT control cells. Histogram represents the mean ± SEM from two independent experiments with 3–5 replicates; ANOVA, P < 0.001, post hoc Fisher's (PLSD) analysis revealed significant differences among (*) groups (1N, 2N, and 4N) for both lines and between (#) the 2N populations. (c) ISH for GFP expression in mTert-GFP adult testis. Dotted lines outline the seminiferous tubules. Arrowheads demarcate the basal layer of spermatogonial stem cells. (Left) Dark field images. (Right) Bright field images. (Magnification: ×10.) (d) Immunohistochemistry for GFP expression in mTert-GFP testis. Arrowhead indicates the basal layer of spermatogonial stem cells. (Magnification: Upper, ×10; Lower, ×40.)

Fig. 3.

Phenotypic Analysis of mTert-GFP expression in HSCs. (a) Increased frequency of LT HSCs and decreased frequency of myeloid progenitors among GFP^hi populations by using multicolor FACS analysis of LT HSCs. Pooled results (mean ± SEM) from two independent experiments; Student's t test indicated. (b) Increased frequency of GFP^hi cells within the LT HSC population compared with the ST HSC population. Pooled results from two independent experiments (mean ± SEM), Student's t test indicated.

Fig. 4.

Functional analysis of mTert-GFP expression in HSCs. (a) FACS scatter plot of BM from a male mTert-GFP mouse demonstrating GFP⁺ cells above the dotted line (set using WT BM). GFP⁺ or GFP⁻ cells were sorted according to the gates shown and transplanted into sublethally irradiated female recipient mice. (b) FACS analysis of BM from female recipient mice 5 months after transplant. GFP⁺ cells were sorted and transplanted into secondary female recipients. (c) FACS analysis of PBCs 5 months after serial BMT confirm serial LT engraftment of GFP⁺ cells. (d) TRAP assay was performed by using isolated GFP⁻ and GFP⁺ BM cells (Fig. 4a) to determine telomerase activity. Heat inactivation samples were used as a negative control. (e) FISH for Y chromosome demonstrated engraftment into both myeloid (Gr-1⁺ and Mac-1⁺) and lymphoid (B220⁺ and CD4⁺) lineages 2 months after serial BMT. (f) FACS analysis of GFP⁺ PBCs 5 months after transplantation confirm GFP in both myeloid (Mac-1⁺) and lymphoid (B220⁺, CD4⁺) lineages at levels comparable to donor animals.

Fig. 5.

GFP expression in intestinal stem cells. Immunohistochemical analysis for GFP and BrdU using serial sections from mTert-GFP mice having undergone BrdU pulse–chase. (a) Staining with anti-GFP antibody, VIP (dark purple) chromagen substrate. (b) Staining with anti-BrdU antibody, DAB (dark brown) chromagen substrate. (Magnification: ×40.)