Development of LT-HSC-Reconstituted Non-Irradiated NBSGW Mice for the Study of Human Hematopoiesis In Vivo

Humanized immune system (HIS) mouse models are useful tools for the in vivo investigation of human hematopoiesis. However, the majority of HIS models currently in use are biased towards lymphocyte development and fail to support long-term multilineage leucocytes and erythrocytes. Those that achieve successful multilineage reconstitution often require preconditioning steps which are expensive, cause animal morbidity, are technically demanding, and poorly reproducible. In this study, we address this challenge by using HSPC-NBSGW mice, in which NOD,B6.SCID IL-2rγ-/-KitW41/W41 (NBSGW) mice are engrafted with human CD133+ hematopoietic stem and progenitor cells (HSPCs) without the need for preconditioning by sublethal irradiation. These HSPCs are enriched in long-term hematopoietic stem cells (LT-HSCs), while NBSGW mice are permissive to human hematopoietic stem cell (HSC) engraftment, thus reducing the cell number required for successful HIS development. B cells reconstitute with the greatest efficiency, including mature B cells capable of class-switching following allogeneic stimulation and, within lymphoid organs and peripheral blood, T cells at a spectrum of stages of maturation. In the thymus, human thymocytes are identified at all major stages of development. Phenotypically distinct subsets of myeloid cells, including dendritic cells and mature monocytes, engraft to a variable degree in the bone marrow and spleen, and circulate in peripheral blood. Finally, we observe human erythrocytes which persist in the periphery at high levels following macrophage clearance. The HSPC-NBSGW model therefore provides a useful platform for the study of human hematological and immunological processes and pathologies.

Programmable microencapsulation for enhanced mesenchymal stem cell persistence and immunomodulation

Mesenchymal stem cell (MSC) therapies demonstrate particular promise in ameliorating diseases of immune dysregulation but are hampered by short in vivo cell persistence and inconsistencies in phenotype. Here, we demonstrate that biomaterial encapsulation into alginate using a microfluidic device could substantially increase in vivo MSC persistence after intravenous (i.v.) injection. A combination of cell cluster formation and subsequent cross-linking with polylysine led to an increase in injected MSC half-life by more than an order of magnitude. These modifications extended persistence even in the presence of innate and adaptive immunity-mediated clearance. Licensing of encapsulated MSCs with inflammatory cytokine pretransplantation increased expression of immunomodulatory-associated genes, and licensed encapsulates promoted repopulation of recipient blood and bone marrow with allogeneic donor cells after sublethal irradiation by a ∼2-fold increase. The ability of microgel encapsulation to sustain MSC survival and increase overall immunomodulatory capacity may be applicable for improving MSC therapies in general.

Distinct Effects of Chondroitin Sulfate on Hematopoietic Cells and the Stromal Microenvironment in Bone Marrow Hematopoiesis

Introduction: Glycosaminoglycans (GAGs), such as heparan sulfate and hyaluronic acid, have been implicated in several hematopoietic processes. GAGs are abundant in the extracellular matrix (ECM) and interact with several cell surface proteins and chemokines. However, the effects of chondroitin sulfate (CS), another species of GAG, in hematopoiesis remain unclear. We examined CS in hematopoiesis by genetically reducing CS in mice by disruption of a gene encoding the rate-limiting CS-synthesizing enzyme N-acetylgalactosaminyltransferase-1 (T1). Methods: T1 knockout (T1KO) mice were generated from the C57BL/6N strain (WT). We evaluated hematopoietic recovery after sublethal irradiation (a 5 Gy dose) to understand the role of CS in hematopoiesis after radiation stress. In addition, we evaluated the effects of each CS on hematopoietic cells and on the stromal microenvironment by creating conditions of CS deficiency in hematopoietic cells or in the stromal microenvironment using hematopoietic stem cell transplantation. In particular, BM cells from WT or T1KO mice were transplanted into 8-10-week-old recipient WT or T1KO mice irradiated at a dose of 9 Gy, and mice were analyzed 5 weeks after transplantation. Furthermore, we examined the role of CS on long-term reconstructive function using a CRU assay in serial transplantation. BM cells from WT or T1KO (CD45.2) mice were transplanted into recipient mice (CD45.1) irradiated at a dose of 9 Gy with BM competitor cells from CD45.1 mice, and PB and BM cell chimerism were analyzed 6 weeks and 12 weeks after transplantation. For serial transplantation, BM cells were collected from recipient mice 12 weeks after transplantation and were transplanted into CD45.1 mice irradiated at a dose of 9 Gy without competitor cells. For evaluating the effect of CS on the stromal microenvironment, BM cells from WT mice were serially transplanted into WT or T1KO recipient mice irradiated at a dose of 9 Gy 12 weeks after transplantation. Results: The amount of CS in BM of T1KO mice was 50-66% of that in WT mice. At steady state, there were no significant differences in the number of PB cells, such as neutrophils, lymphocytes, RBCs and platelets, and total BM cells in T1KO and WT mice. T1KO mice had a significantly higher number of BM LSK cells compared to that of WT mice (WT: 0.213 ± 0.044%; T1KO: 0.282 ± 0.046%, p < 0.01). The corresponding number of CFU-GM of BM cells was also higher in the T1KO mice group (WT: 29.6 ± 3.60; T1KO: 45.4 ± 2.37, p < 0.01). However, hematopoietic recovery (PB cells, total BM cells, and LSK cells) after sublethal irradiation was significantly delayed in T1KO mice. CS deficiency in hematopoietic cells resulted in a lower number of LSK cells compared to that of WT hematopoietic cells after transplantation (WT: 0.176 ± 0.078%; T1KO: 0.131 ± 0.046% p < 0.05). Conversely, no significant difference was observed in mice with CS-reduced stroma. To reveal the effect of CS in hematopoietic cells on long-term reconstructive function, we evaluated the chimerism of PB Gr1+CD11b+cells, B220+ cells, CD3+ cells, and BM LSK cells by a CRU assay. In the first transplantation, there were no significant differences in short-term reconstitution (after 6 weeks) and long-term reconstitution (after 12 weeks). In the second transplantation, hematopoietic cells derived from T1KO mice had lower chimerism in all PB cell lineages. Next, we evaluated the role of CS on the stromal microenvironment by serial transplantation. In the first transplantation, there were no significant differences between PB and BM cells. In the second transplantation, the proportion of BM LSK cells was higher in T1KO recipient mice (CS deficiency in the stroma). Conclusion: CS may have an important role in hematopoiesis. CS in hematopoietic cells and the stromal microenvironment had different effects on BM hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Vasopressin stimulates the proliferation and differentiation of red blood cell precursors and improves recovery from anemia

Arginine vasopressin (AVP) made by hypothalamic neurons is released into the circulation to stimulate water resorption by the kidneys and restore water balance after blood loss. Patients who lack this antidiuretic hormone suffer from central diabetes insipidus. We observed that many of these patients were anemic and asked whether AVP might play a role in red blood cell (RBC) production. We found that all three AVP receptors are expressed in human and mouse hematopoietic stem and progenitor cells. The AVPR1B appears to play the most important role in regulating erythropoiesis in both human and mouse cells. AVP increases phosphorylation of signal transducer and activator of transcription 5, as erythropoietin (EPO) does. After sublethal irradiation, AVP-deficient Brattleboro rats showed delayed recovery of RBC numbers compared to control rats. In mouse models of anemia (induced by bleeding, irradiation, or increased destruction of circulating RBCs), AVP increased the number of circulating RBCs independently of EPO. In these models, AVP appears to jump-start peripheral blood cell replenishment until EPO can take over. We suggest that specific AVPR1B agonists might be used to induce fast RBC production after bleeding, drug toxicity, or chemotherapy.

EMSA Eritin Drives Expansion of Regulatory T Cells and Promotes T Cells Differentiation in Irradiated Mice

Sublethal irradiation therapy in cancer treatment causes generalized immunosuppression, which results in a range of DNA damage. We examined the significance of a polyherbal medicine called “EMSA Eritin” on immunological responses in sublethally irradiated mice focusing on the involvement of Treg, naïve T cell, and also the development and differentiation of T cells in thymus. Normal BALB/c mice were sublethally irradiated with dose of 600 rad. The irradiated mice were then orally administered by EMSA Eritin once a day at different doses: 1.04, 3.12, 9.37 mg/g body weight. The treatment was performed for 14 days. On day 15, immunological responses were observed by analyzing the status of Treg and differentiation of T cells in thymus. The administration of EMSA Eritin to irradiated mice resulted in a significant increase of pre T cells, Treg cells, and naïve T cells, which in general could maintain and normalize healthy condition in mice.