New genetic tools for the in vivo study of hematopoietic stem cell function

Abstract

The production of blood cells is dependent on the activity of a rare stem cell population that normally resides in the bone marrow (BM) of the organism. These hematopoietic stem cells (HSCs) have the ability to both self-renew and differentiate, ensuring this lifelong hematopoiesis. Determining the regulation of HSC functions should thus provide critical insight to advancing regenerative medicine. Until quite recently, HSCs were primarily studied using in vitro studies and transplantations into immunodeficient hosts. Indeed, the definition of a bona fide HSC is its ability to reconstitute lymphopenic hosts. In this review, we discuss the development of novel, HSC-specific genetic reporter systems that enable the prospective identification of HSCs and the study of their functions in the absence of transplantation. Coupled with additional technological advances, these studies are now defining the fundamental properties of HSCs in vivo. Furthermore, complex cellular and molecular mechanisms that regulate HSC dormancy, self-renewal, and differentiation are being identified and further dissected. These novel reporter systems represent a major technological advance for the stem cell field and allow new questions to be addressed.

Figure 1. Use of KI and Tg approaches to generate HSC-specific genetic reporter animals

Both strategies use regulatory elements of a gene of interest to drive expression of a reporter cassette. This cassette can be inserted 5′ (as depicted above, immediately after the ATG start codon) or 3′ of the coding sequence. (A) KI animals can be generated by targeting of ES cells or using the CRISPR/Cas9 system. To modify ES cells, a targeting vector that encodes reporter and antibiotic resistance cassettes flanked by arms of homologous sequence both directly 5′ and 3′ of the targeted locus is electroporated into ES cells. These flanking arms facilitate homologous recombination between the vector insert and ES cell genomic DNA, and ES cells are selected for the appropriate antibiotic resistance and screened for targeting vector incorporation. Correctly targeted ES cells are injected into a blastocyst of a different genetic background, which is transferred to a pseudopregnant female. ES cell contribution to the resulting pup is visualized by the different coat colors associated with the genetic backgrounds of the ES cells and blastocyst. Chimeric animals must be further bred to confirm germline contribution from the ES cells. Alternatively, KI animals can be generated by the direct injection of mRNA encoding the Cas9 nuclease, an sgRNA that directs DNA binding of Cas9, and a donor template vector encoding the reporter cassette into a fertilized egg. DSB mediated by Cas9 can be resolved by HDR, leading to incorporation of the reporter cassette at the desired genomic locus. The zygote is transferred to a pseudopregnant female, and the resulting progeny are screened for the correct genotype. (B) BAC Tg animals are made in a similar manner to KI animals made using CRISPR/Cas9. A reporter cassette is introduced into a BAC clone that encodes the locus of interest by recombineering. The targeted BAC DNA becomes randomly incorporated into the genomic DNA of a fertilized egg, which is then transferred to a pseudopregnant female mouse. Resulting offspring are genotyped for the Tg.

Figure 2. Studying HSC functions in vivo using genetic reporter systems

HSC-specific reporters can be combined with other inducible genetic mouse strains to study in vivo functions of HSCs. (A) In vivo HSC contribution to hematopoiesis can be studied by lineage tracing. In the absence of tamoxifen, the CreERT2 recombinase is retained in the cytoplasm and a LoxP-flanked Stop cassette encoding repeating polyadenylation signals prevents expression of the fluorescent reporter (e.g. tdTomato) in cells that express the HSC reporter. Tamoxifen treatment enables CreERT2 translocation to the nucleus where it mediates excision of the Stop cassette. This results in irreversible expression of tdTomato, and any daughter cell that arises from the labeled cell will also express tdTomato. The contribution of the labeled cell to the production of other cell types is estimated by measuring the fraction of tdTomato⁺ cells over time. (B) Dormant HSCs can be identified in vivo by the ability to stably retain human histone H2B-GFP fluorescent label. A TRE regulates the expression of H2B-GFP, and this system can function as a “Tet-off” or “Tet-on” system. In Tet-off systems, H2B-GFP is induced in cells that express the HSC-specific tTA. In the presence of the tetracycline analog doxycycline (dox) the de novo synthesis of H2B-GFP is inhibited. The H2B-GFP protein is highly stable but becomes diluted upon proliferation of labeled cells over time. Cells that retain the fluorescent label over a long period of continuous dox treatment are considered stable label retaining cells. In a Tet-on system, H2B-GFP expression is induced in cells that express the HSC-specific reverse tetracycline transactivator (rtTA) only upon treatment with dox. Once complete labeling of the population of interest is confirmed, dox is removed and cells are assessed for label retention over time.