Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis (CH) is a condition which is frequently observed in elderly individuals and occurs as a result of an expanded hematopoietic stem and progenitor cell (HSPC) clone carrying a genetic mutation¹. It has been suggested that by the age of 50, most individuals will have acquired an average of five exonic mutations in each HSPC², but most of these mutations will result in little or no phenotypic consequences to the individual. However, if by chance one of these mutations confers a competitive advantage to the HSPC—such as by promoting it’s proliferation, self-renewal, survival, or some combination of these—this may lead to the preferential expansion of the mutant clone relative to the other HSPCs. As a result, the mutation will increasingly spread through the hematopoietic system as the mutated HSPC gives rise to mature blood cells, leading to a distinct population of mutated cells within the peripheral blood. While mutations in dozens of different candidate driver genes have been associated with clonal events within the hematopoietic system, among these, mutations in DNA methyltransferase 3 alpha (DNMT3A) and ten eleven translocation 2 (TET2) are the most prevalent³. Several epidemiological studies have found that individuals who carry these genetic mutations have a significantly higher risk of cardiovascular disease (CVD), stroke, and all-causal mortality^3,4,5,6,7. While these studies have identified that an association exists between CH and increased incidence of CVD and stroke, we do not know whether this relationship is causal or a shared epiphenomenon with the aging process. To gain a better understanding of this association, proper animal models that correctly recapitulate the human condition of CH are required.

Several CH animal models have been established by our group and others using zebrafish, mice, and non-human primates^{8,9,10,11,12,13,14}. These models often use hematopoietic reconstitution methods by transplantation of genetically modified cells, sometimes using Cre-lox recombination or the CRISPR system. This approach allows for the analysis of a specific gene mutation in hematopoietic cells to assess how it contributes to disease development. In addition, these models often employ congenic or reporter cells to distinguish the effects of mutant cells from normal or wild-type cells. In many cases, a pre-conditioning regimen is required to successfully engraft donor hematopoietic stem cells.

Currently, the transplantation of bone marrow to recipient mice can be divided into two main categories: 1) myeloablative conditioning and 2) non-conditioned transplantation. Myeloablative conditioning can be achieved by one of two methods, namely, total body irradiation (TBI) or chemotherapy¹⁵. TBI is carried out by subjecting the recipient to a lethal dose of gamma or X-ray irradiation, generating DNA breaks or cross-links within rapidly dividing cells, rendering them irreparable¹⁶. Busulfan and cyclophosphamide are two commonly used chemotherapy drugs that disrupt the hematopoietic niche and similarly cause DNA damage to rapidly dividing cells. The net result of myeloablative preconditioning is apoptosis of hematopoietic cells, which destroys the recipient’s hematopoietic system. This strategy not only allows for the successful engraftment of the donor HSPCs, but can also prevent graft rejection by suppressing the recipient’s immune system. However, myeloablative preconditioning has severe side effects such as damage to tissues and organs and their resident immune cells as well as destruction of the native bone marrow niche¹⁷. Therefore, alternative methods have been proposed to overcome these undesirable side effects, particularly in regard to damage to the organs of interest. These methods include shielded irradiation of recipient mice and the adoptive BMT to non-conditioned mice^9,17. Shielding the thorax, abdominal cavity, head or other regions from irradiation by the placement of a lead barriers keeps tissues of interest protected from the damaging effects of irradiation and maintains their resident immune cell population. On the other hand, the adoptive BMT of HSPCs to non-conditioned mice has an additional advantage because it preserves the native hematopoietic niche. In this manuscript, we describe the protocols and results of HSPC engraftment after several transplantation regimens in mice, specifically the delivery of HSPC to TBI mice, to mice partially shielded from irradiation, and to non-conditioned mice. The overall goal is to help researchers understand the different physiological effects of each method as well as how they affect experimental outcomes in the setting of CH and cardiovascular disease.