Second bone marrow transplantation into regenerating hematopoiesis enhances reconstitution of immune system.

In bone marrow transplantation (BMT), hematopoiesis-reconstituting cells are introduced following myeloablative treatment, which eradicates existing hematopoietic cells and disrupts stroma within the hematopoietic tissue. Both hematopoietic cells and stroma then undergo regeneration. Our study compares the outcomes of a second BMT administered to mice shortly after myeloablative treatment and the first BMT, with those of a second BMT administered to mice experiencing robust hematopoietic regeneration after the initial transplant. We evaluated the efficacy of the second BMT in terms of engraftment efficiency, types of generated blood cells, and longevity of function. Our findings show that regenerating hematopoiesis readily accommodates newly transplanted stem cells, including those endowed with a robust capacity for generating B and T cells. Importantly, our investigation uncovered a window for preferential engraftment of transplanted stem cells coinciding with the resumption of blood cell production. Repeated BMT could intensify hematopoiesis reconstitution and enable therapeutic administration of genetically modified autologous stem cells.

Hematopoiesis Remains Permissive to Bone Marrow Transplantation After Expansion of Progenitors and Resumption of Blood Cell Production.

The immense regenerative power of hematopoietic tissue stems from the activation of the immature stem cells and the progenitor cells. After partial damage, hematopoiesis is reconstituted through a period of intense regeneration when blood cell production originates from erythro-myeloid progenitors in the virtual absence of stem cells. Since the damaged hematopoiesis can also be reconstituted from transplanted hematopoietic cells, we asked whether this also leads to the transient state when activated progenitors initially execute blood cell production. We first showed that the early reconstitution of hematopoiesis from transplanted cells gives rise to extended populations of developmentally advanced but altered progenitor cells, similar to those previously identified in the bone marrow regenerating from endogenous cells. We then identified the cells that give rise to these progenitors after transplantation as LSK CD48- cells. In the submyeloablative irradiated host mice, the transplanted LSK CD48- cells preferably colonized the spleen. Unlike the endogenous hematopoiesis reconstituting cells, the transplanted whole bone marrow cells and sorted LSK CD48- cells had greater potential to differentiate to B-lymphopoiesis. Separate transplantation of the CD150- and CD150+ subsets of LSK CD48- cells suggested that CD150- cells had a greater preference to B-lymphopoiesis than CD150+ cells. In the intensively regenerating hematopoiesis, the CD71/Sca-1 plot of immature murine hematopoietic cells revealed that the expanded populations of altered myeloid progenitors were highly variable in the different places of hematopoietic tissues. This high variability is likely caused by the heterogeneity of the hematopoiesis supporting stroma. Lastly, we demonstrate that during the period when active hematopoiesis resumes from transplanted cells, the hematopoietic tissues still remain highly permissive for further engraftment of transplanted cells, particularly the stem cells. Thus, these results provide a rationale for the transplantation of the hematopoietic stem cells in successive doses that could be used to boost the transplantation outcome.

Altered Erythro-Myeloid Progenitor Cells Are Highly Expanded in Intensively Regenerating Hematopoiesis.

Regeneration of severely damaged adult tissues is currently only partially understood. Hematopoietic tissue provides a unique opportunity to study tissue regeneration due to its well established steady-state structure and function, easy accessibility, well established research methods, and the well-defined embryonic, fetal, and adult stages of development. Embryonic/fetal liver hematopoiesis and adult hematopoiesis recovering from damage share the need to expand populations of progenitors and stem cells in parallel with increasing production of mature blood cells. In the present study, we analyzed adult hematopoiesis in mice subjected to a submyeloablative dose (6 Gy) of gamma radiation and targeted the period of regeneration characterized by massive production of mature blood cells along with ongoing expansion of immature hematopoietic cells. We uncovered significantly expanded populations of developmentally advanced erythroid and myeloid progenitors with significantly altered immunophenotype. Their population expansion does not require erythropoietin stimulation but requires the SCF/c-Kit receptor signaling. Regenerating hematopoiesis significantly differs from the expanding hematopoiesis in the fetal liver but we find some similarities between the regenerating hematopoiesis and the early embryonic definitive hematopoiesis. These are in (1) the concomitant population expansion of myeloid progenitors and increasing production of myeloid blood cells (2) performing these tasks despite the severely reduced transplantation capacity of the hematopoietic tissues, and (3) the expression of CD16/32 in most progenitors. Our data thus provide a novel insight into tissue regeneration by suggesting that cells other than stem cells and multipotent progenitors can be of fundamental importance for the rapid recovery of tissue function.

Cell cycle and differentiation of Sca-1+ and Sca-1- hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analyzed the cell cycle and cell production rate in HSPCs in murine hematopoiesis. The labeling of DNA-synthesizing cells by two thymidine analogues, optimized for in-vivo use, enabled determination of the cell cycle flow rate into G2-phase, the duration of S-phase and the average cell cycle time in Sca-1+ and Sca-1- HSPCs. Determination of cells with 2n DNA content labeled in preceding S-phase was then used to establish the cell flow rates in G1-phase. Our measurements revealed a significant difference in how Sca-1+ and Sca-1- myeloid progenitors self-renew and differentiate. Division of the Sca-1+ progenitors led to loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. Sca-1- cells arising from cell division entered a new round of the cell cycle, corresponding to symmetric self-renewing cell division. The novel data also enabled the estimation of the cell production rates in Sca-1+ and in three subtypes of Sca-1- HSPCs and revealed Sca-1 negative cells as the major amplification stage in the blood cell development.

Stem Cell Defect in Ubiquitin-Green Fluorescent Protein Mice Facilitates Engraftment of Lymphoid-Primed Hematopoietic Stem Cells.

Transgenic mice expressing green fluorescent protein (GFP) are useful in transplantation experiments. When we used ubiquitin-GFP (UBC-GFP) transgenic mice to study the availability of niches for transplanted hematopoietic stem and progenitor cells, the results were strikingly different from the corresponding experiments that used congenic mice polymorphic in the CD45 antigen. Analysis of these unexpected results revealed that the hematopoiesis of UBC-GFP mice was outcompeted by the hematopoiesis of wild-type (WT) mice. Importantly, UBC-GFP mice engrafted the transplanted bone marrow of WT mice without conditioning. There was a significant bias toward lymphopoiesis in the WT branch of chimeric UBC-GFP/WT hematopoiesis. A fraction of immature Sca-1+ cells in the spleen of UBC-GFP mice expressed GFP at a very high level. The chimeric hematopoiesis was stable in the long term and also after transplantation to secondary recipient mice. The article thus identifies a specific defect in the hematopoiesis of UBC-GFP transgenic mice that compromises the lymphoid-primed hematopoietic stem cells in the bone marrow and spleen. Stem Cells 2018;36:1237-1248.

Low c-Kit Expression Level Induced by Stem Cell Factor Does Not Compromise Transplantation of Hematopoietic Stem Cells.

The c-Kit expression level is decreased in regenerating bone marrow, and such bone marrow performs poorly when co-transplanted with normal bone marrow. We asked whether diminished numbers of c-Kit receptors on hematopoietic stem and progenitor cells (HSPCs) after their internalization induced by the binding of the cytokine stem cell factor (SCF) would jeopardize transplantability of HSPCs. We used a battery of functional assays to evaluate the capacity of HSPCs with markedly different c-Kit expression levels to be transplanted. Surprisingly, our experiments testing the homing of transplanted HSPCs to bone marrow of recipient mice and their short-term and long-term engraftment did not reveal any defects in HSPCs with severely reduced numbers of c-Kit receptor molecules. This unexpected result can be ascribed to the fact that HSPCs exposed to SCF replace the consumed c-Kit receptors rapidly. This article demonstrates that exposure of HSPCs to SCF and diminished number of c-Kit receptors in their cell membranes do not compromise the capacity of HSPCs to reconstitute damaged hematopoietic tissue.

Allometry of sexual size dimorphism in domestic dog.

BACKGROUND: The tendency for male-larger sexual size dimorphism (SSD) to scale with body size - a pattern termed Rensch's rule - has been empirically supported in many animal lineages. Nevertheless, its theoretical elucidation is a subject of debate. Here, we exploited the extreme morphological variability of domestic dog (Canis familiaris) to gain insights into evolutionary causes of this rule. METHODOLOGY/PRINCIPAL FINDINGS: We studied SSD and its allometry among 74 breeds ranging in height from less than 19 cm in Chihuahua to about 84 cm in Irish wolfhound. In total, the dataset included 6,221 individuals. We demonstrate that most dog breeds are male-larger, and SSD in large breeds is comparable to SSD of their wolf ancestor. Among breeds, SSD becomes smaller with decreasing body size. The smallest breeds are nearly monomorphic. CONCLUSIONS/SIGNIFICANCE: SSD among dog breeds follows the pattern consistent with Rensch's rule. The variability of body size and corresponding changes in SSD among breeds of a domestic animal shaped by artificial selection can help to better understand processes leading to emergence of Rensch's rule.

Hematopoietic stem cells survive circulation arrest and reconstitute hematopoiesis in myeloablated mice.

Hematopoietic stem and progenitor cells (HSPC) for bone marrow transplantation are currently obtained directly from living voluntary donors or from cord blood units. However, a suitable donor is not always found. Because HSPC are known for their relative resistance to hypoxia, using an experimental murine model, we explored cadaveric bone marrow (BM) as their alternative source. After donor mice were sacrificed, BM was left in intact femurs at 37°C, 20°C, or 4°C under ischemic conditions, resulting in combined oxygen and metabolic substrate shortage and the accumulation of metabolic waste products. BM cells were harvested after a set time period ranging from 0 to 48 hours. To determine the impact of delayed harvesting on the transplantability of HSPC, a competitive repopulation assay using a murine Ly5.1/Ly5.2 congenic model in 2 different settings was used: after submyeloablative (6 Gy) or myeloablative (9 Gy) total-body irradiation, Ly5.2 hosts received cadaveric Ly5.1 cells or a mixture of cadaveric Ly5.1 cells and fresh Ly5.2 cells in a 1:1 ratio. Chimerism resulting from cadaveric donor cells, followed up to 6 months after transplantation, proved that the long-term repopulation ability of HSPC was fully preserved for 2 hours, 6 hours, and 12 hours at 37°C, 20°C, and 4°C of ischemia, respectively. A colony-forming unit-spleen (CFU-S) clonogenic assay revealed a higher sensitivity of proliferating hematopoietic progenitors to ischemia compared to repopulating cells (STRC and LTRC). Flow cytometry analysis of apoptosis in cadaveric BM demonstrated that the LSK (Lin(low)Sca-1(+)c-Kit(+)) subpopulation, enriched in HSPC, contained less apoptotic and dead cells than the BM as a whole. Furthermore, the number of LSK SLAM (CD150(+)CD48(-)) and LSK SP (side population) cells (fractions highly enriched in hematopoietic stem cells) decreased in parallel with BM transplantability. As well as cadaveric BM cells, we also tested the transplantability and survival of BM cells after storage in a suspension in vitro without specific hematopoietic growth factors. HSPC did not display any decrease in transplantability after 2 days of storage at 37°C or 4 days at 4°C. A higher sensitivity of progenitors to unfavorable conditions was observed again using CFU-S and granulocyte macrophage-colony forming cell (GM-CFC) assays, especially at 37°C. This paper shows that HSPC survive the cessation of circulation for a considerable time and maintain their engraftment potential. This time is significantly extended with in vitro storage compared to the cadaveric BM.