Differentiation Capability And Adhesion Molecular Expression Of Bone Marrow Mesenchymal Stem Cells Derived From Patients Who Diagnosed As Acute Myeloid Leukemia And Non-Hodgkin Lymphoma

In the pathological research of leukemia, there is an emerging evidence suggesting that apart from gene mutations in hematopoietic stem cells, the bone marrow microenvironment also plays an important role in the proliferation, differentiation and migration of malignent stem cells. It was suggested that specific niche is necessary for the maintenance of hematopoietic stem cells (HSC). If hematopoietic stem cells move away from the niche or the microenvironment changes, stem cells will tend to proliferate and differentiate into mature cell types. The niche is comprised of stromal cells, such as mesenchymal stem cells, osteoblasts, adipocyte, vascular endothelial cells and fibroblast, and extracellular matrix. Many components are reported to be able to regulate HSC function. For example, osteoblastic cells can control the niche size by direct cell-cell contact and releasing of regulatory factors. Recently, MSCs are also identified to be an essential HSC niche component, and in vivo MSCs depletion reduces HSC content and haematopoietic progenitors bone marrow homing. MSCs can be further differentiated into osteoblasts and adipocyte, thus the differentiation ability of MSCs also influences the niche function. Apart from proliferation and differentiation, the bone marrow homing capacity of haematopoietic progenitors into bone marrow is important for clinical stem cell engraftment. The expression of adhesion molecule on niche component cells is thought to be related to HSC homing, and of course MSCs are involved. However, there is few report about the characteration, differentiation and adhesion ability of MSCs from leukemia patients. We examined the adipogenic and osteogenic differentiation capability of bone marrow mesenchymal stem cells in patients who diagnosed as acute myeloid leukemia, non-Hodgkin lymphoma and normal control. Expression of related genes were determined by Real-time PCR, providing clinical data to further explorer the role of MSCs in hematopoietic stem cell niche maintenance and leukemia pathological research.Parti. Comparasion of the character and differentiation capability of BM-MSCs from acute myeloid leukemia and non-Hodgkin lymphoma patientsObjective:Compare the character and adipogenic, osteogenic differentiation capability of bone marrow mesenchymal stem cells in patients of acute myeloid leukemia non-Hodgkin lymphoma and analyze the expression of marker genes to explorer its function in HSC niche and AML pathology.Method:Two-point-five ml bone marrow was extracted from patients into EDTA tube with5ML PBS. Then add3.5ML Ficoll extraction liquid, centrifugal2000rpm for20min. Absorb single nucleus layer and culture them into spindle morphology. Then MSC positive marker CD105、CD73、CD90and negative marker CD45were identified by flow cytometry. Using conditional medium to adipogenic and osteogenic differentiation, we extracted mRNA before and at day3,7and utiliezd real-time PCR method to analyze lipid gene LPL and osteogenic gene OPN. At day14, lipid cell and osteoblasts were stained by specific method.Results:MSCs from patients usually showed spindle morphology and there were a few round floating cells. However, primary MSCs from AML patients showed aberrant morphology compared with control MSCs, illustrated by more branches and more round cells. After4generations, three groups of cells showed similar spindle morphology. Flow cytometry identified MSC positive marker CD105、CD73、CD90and negative marker CD45, confirming these cells are MSCs. After adipogenic differentiation, lipid drops appeared early and more in AML-MSCs later. At day14, oil red staining showed no difference among three groups. LPL (Lipoprotein lipase) is a commonly used marker gene for lipid cells. RT-PCR results showed LPL’s expression significantly increased after3days and7days of adipogenic differentiation. Before adipogenic differentiation, lipid marker gene LPL from AML-MSCs expressed significantly higher than NHL-MSCs. After adipogenic differentiation, LPL’s expression showed no significantly difference among three groups. On the other hand, after osteogenic differentiation, AML group showed a weaker staining at day14, but osteogenic gene OPN’s expression showed no change and no difference among three groups.Conclusion:Primary MSCs from AML patients show aberrant morphology compared with control MSCs. Before adipogenic differentiation, lipid marker gene LPL from AML-MSCs express significantly higher than NHL-MSCs and Control-MSCs. After adipogenic differentiation, lipid drops appear early and more in AML-MSCs. On the other hand, after osteogenic differentiation, AML-MSCs show less staining for osteoblasts.Part2. Comparison of adhesion molecular expression of BM-MSCs from acute myeloid leukemia and non-Hodgkin lymphoma patientsObjective:Compare the adhesion genes expression of bone marrow mesenchymal stem cells in patients of acute myeloid leukemia, non-Hodgkin lymphoma, Control group and the expression after differentiation to explorer its function in HSC niche and AML pathology.Method:Extract2.5ML bone marrow from patients into EDTA tube with5ML PBS. Then add3.5ML Ficoll extraction liquid, centrifugal2000rpm for20min. Absorb single nucleus layer and culture into spindle morphology. Then use flow cytometry to identify MSC positive marker CD105、CD73、CD90and negative marker CD45. Using conditional medium to adipogenic and osteogenic differentiation, extract mRNA before and at day3,7and using real-time PCR to analyze adhesion molecular E-Cadherin and N-Cadherin.Results:Before differentiation, N-Cadherin and E-Cadherin expression of MSCs from AML patients show no significant difference with those from NHL patients and control. After adipogenic differentiation, there is a significant down regulation in E-Cadherin expression of MSCs from AML patients at day3and day7. Thus, E-Cadherin shows a lower expression in AML-MSCs than Control-MSCs at day3. On the other hand, after osteogenic differentiation, E-Cadherin’s expression is down regulated in NHL-MSCs and Control-MSCs, which leads to E-Cadherin’s higher expression in AML-MSCs than Control-MSCs at day7. Besides, MSCs in patients of AML patients show similar N-Cadherin expression with patients of NHL and control people.Conclusion:After adipogenic differentiation, adhesion molecular E-Cadherin’s but not N-cadherin’s expression is down regulated in AML-MSCs, which leads to E-Cadherin’s lower expression in AML-MSCs than Control-MSCs at day3. On the other hand, after osteogenic differentiation, E-Cadherin’s expression is down regulated in NHL-MSCs and Control-MSCs, which leads to E-Cadherin’s higher expression in AML-MSCs than Control-MSCs at day7.In conclusion, our study indicated that primary MSCs from AML patients show aberrant morphology compared with control MSCs. Before adipogenic differentiation, lipid marker gene LPL from AML-MSCs express significantly higher than NHL-MSCs and Control-MSCs. After adipogenic differentiation, lipid drops appear early and more in AML-MSCs, and adhesion molecular E-Cadherin’s but not N-cadherin’s expression is down regulated in AML-MSCs, which leads to E-Cadherin’s lower expression in AML-MSCs than Control-MSCs at day3. On the other hand, after osteogenic differentiation, AML-MSCs show less staining for osteoblasts, E-Cadherin’s expression is down regulated in NHL-MSCs and Control-MSCs, which leads to E-Cadherin’s higher expression in AML-MSCs than Control-MSCs at day7.