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.