CD34+ Cobblestone Area-Forming Cells Are a Valuable Indicator to Screen the Soluble Inhibitors of Hematopoiesis in the Serum of Aplastic Anemia and MDS Patients.

Abstract 1439

Poster Board I-462

We have previously shown that cobblestone area (CA) could be observed underneath human stromal cells 2 weeks after coculture in serum free condition. However, it was difficult to evaluate 5 week CA-forming cells (CAFC) which is a surrogate indicator of hematopoietic stem cells because human stromal cells could not survive more than 4 weeks in a serum free media, X-VIVO10. In the present study, we optimized serum free media, combination of cytokines and chemical agents such as stem cell factor (SCF), thrombopoietin (TPO), flt3 ligand (FL) with or without delta like protein 4 (DLL4) and GSK inhibitor. In addition, we added the serum derived from aplastic anemia and MDS patients in this coculture system and evaluated the effect of serum on in vitro hematopoiesis. We found that certain serum free media could maintain the layer of human stromal cells more than 8 weeks. By using this serum free media, bone marrow (BM) CD34+ cells could be cocultured for 8 weeks without disruption of the human stromal layer. Five week CAFC could be observed in all condition of cytokine combination with or without chemical inhibitor. However, because cytoplasmic appearance of cells in some CAs is quite irregular without DLL4 or GSK inhibitor, we conducted immunohistochemical staining by using FITC-labeled anti human CD34, CD11b or CD33 antibodies. Surprisingly, we found that CD34+CAFCs were exclusively observed in the presence of DLL4 or GSK inhibitor. Otherwise, CAs was composed of CD11b+/CD45+ hematopoietic cells. Moreover, NOD/SCID repopulating cells were detected in the coculture containing CD34+CAFCs, suggesting that CD34+CAFCs were a valuable indicator of hematopoietic stem cells (HSCs). Using CD34+CAFCs as a surrogate maker of HSCs, we assessed whether the serum derived from patients of aplastic anemia and MDS could inhibit formation of CD34+CAFC. Notably, some of patient's serum could reduce the number of CD34+CAFCs. These results indicated the possibility that CD34+CAFCs underneath human stromal layer could be useful for screening certain soluble factors to inhibit hematopoiesis in aplastic anemia and MDS.