JDM  Vol.1 No.4 , November 2011
Bone marrow increases human islets insulin positive cells in co-culture: quantification with flow cytometry
Abstract: We have previously demonstrated that allogeneic human bone marrow (BM) supports human islet function and longevity in vitro. We hypothesize that BM supporting human islets may include to increase β-cell in cultured islets. In this study, we developed a method to quantify insulin-producing β cells from cultured islets by using immunofluorescent staining and flow cytometry analysis to explore this possibility. The results show that human islets cocultured with BM for 39 days contained a significantly higher number of insulin-positive β cells (42.3% ± 4.5%) compared to the islet-only cultures (1.15% ± 0.78%), and increased insulin release levels evaluated by ELISA is consistent with increased β cells in same culture condition. Human islet culture with BM significantly increase β-cells while islet only culture lost β-cells in same culture period supports the possibility of BM increasing β-cells in cultured islets.
Cite this paper: nullWang, Z. , Xiong, F. , Hassani, M. , Luo, J. and Luo, L. (2011) Bone marrow increases human islets insulin positive cells in co-culture: quantification with flow cytometry. Journal of Diabetes Mellitus, 1, 109-117. doi: 10.4236/jdm.2011.14015.

[1]   Shapiro, A.M., Lakey, J.R., Ryan, E.A., et al. (2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. The New England Journal of Medicine, 43, 230-238. doi:10.1056/NEJM200007273430401

[2]   Shapiro, A.M., Ricordi, C., Hering, B.J., et al. (2006) International trial of the Edmonton protocol for islet transplantation. The New England Journal of Medicine, 355, 1318-1330. doi:10.1056/NEJMoa061267

[3]   Shapiro, A.M.J., Ryan, E.A. and Lakey, J.R.T. (2001) Islet cell transplantation. The Lancet, 358, 07034-07039. doi:10.1016/S0140-6736(01)07034-9

[4]   Shapiro, A.M.J., Ryan, E.A. and Lakey, J.R.T. (2001) Clinical islet transplant—State of the art. Transplantation Proceedings, 33, 3502-3503. doi:10.1016/S0041-1345(01)02416-2

[5]   Matsumoto, S., Goel, S., Qualley, S., Strong, D.M. and Reems, J.A. (2003) A comparative evaluation of culture conditions for short-term maintenance (<24 hr) of human islets isolated using the Edmonton protocol. Cell and Tissue Banking, 4, 85-93. doi:10.1023/B:CATB.0000007043.15164.8a

[6]   Paraskevas, S., Maysinger, D., Wang, R., Duguid, T.P. and Rosenberg, L. (2000) Cell loss in isolated human islets occurs by apoptosis. Pancreas, 20, 270-276. doi:10.1097/00006676-200004000-00008

[7]   Noguchi, H. (2007) Activation of c-Jun NH2-terminal kinase during islet isolation. Endocrine Journal, 54, 169-176. doi:10.1507/endocrj.KR-87

[8]   Iwanaga, Y., Sutherland, D.E., Harmon, J.V. and Papas, K.K. (2008) Pancreas preservation for pancreas and islet transplantation. Current Opinion in Organ Transplantation, 13, 135-141. doi:10.1097/MOT.0b013e3282f63942

[9]   Luo, L., Badiavas, E., Luo, J.Z. and Maizel, A. (2007) Allogeneic bone marrow supports human islet beta cell survival and function over six months. Biochemical and Biophysical Research Communications, 361, 859-864. doi:10.1016/j.bbrc.2007.07.105

[10]   Sai, P. and Maurel, C. (1983) Enzyme-linked immunosorbent assay for quantitation of islet cell antibodies. Journal of Immunological Methods, 64, 189-198.

[11]   Kuglin, B., Gries, F.A. and Kolb, H. (1988) Evidence of IgG autoantibodies against human proinsulin in patients with IDDM before insulin treatment. Diabetes, 37, 130-132. doi:10.2337/diabetes.37.1.130

[12]   Sodoyez, J.C., Koch, M., Lemaire, I., et al. (1991) Influence of affinity of antibodies upon their detection by liquid phase radiobinding assay and solid phase enzyme linked immunosorbent assay. Demonstration using monoclonal antibodies raised against rDNA human proinsulin. Diabetologia, 34, 463-468. doi:10.1007/BF00403281

[13]   Hartling, S.G., Dinesen, B., Kappelgard, A.M., Faber, O.K. and Binder, C. (1986) ELISA for human proinsulin. Clinica Chimica Acta: International Journal of Clinical Chemistry, 156, 289-297.

[14]   Ravazzola, M., Perrelet, A., Roth, J. and Orci, L. (1981) Insulin immunoreactive sites demonstrated in the Golgi apparatus of pancreatic B cells. Proceedings of the National Academy of Sciences of the United States of America, 78, 5661-5664. doi:10.1073/pnas.78.9.5661

[15]   Reddy, S.N., Bibby, N.J. and Elliott, R.B. (1985) Cellular distribution of insulin, glucagon, pancreatic polypeptide hormone and somatostatin in the fetal and adult pancreas of the guinea pig: A comparative immunohistochemical study. European Journal of Cell Biology, 38, 301-305.

[16]   Lukowiak, B., Vandewalle, B., Riachy, R., et al. (2001) Identification and purification of functional human beta- cells by a new specific zinc-fluorescent probe. Journal of Histochemistry and Cytochemistry, 49, 519-528. doi:10.1177/002215540104900412

[17]   Fernandez, L.A., Hatch, E.W., Armann, B., et al. (2005) Validation of large particle flow cytometry for the analysis and sorting of intact pancreatic islets. Transplantation, 80, 729-737. doi:10.1097/

[18]   Jayaraman, S. (2008) A novel method for the detection of viable human pancreatic beta cells by flow cytometry using fluorophores that selectively detect labile zinc, mitochondrial membrane potential and protein thiols. Cytometry Part A, 73, 615-625. doi:10.1002/cyto.a.20560

[19]   Dubben, S., Honscheid, A., Winkler, K., Rink, L. and Haase, H. (2010) Cellular zinc homeostasis is a regulator in monocyte differentiation of HL-60 cells by 1 alpha, 25-dihydroxyvitamin D3. Journal of Leukocyte Biology, 87, 833-844. doi:10.1189/jlb.0409241

[20]   Kaltenberg, J., Plum, L.M., Ober-Blobaum, J.L., Honscheid, A., Rink, L. and Haase, H. (2010) Zinc signals promote IL-2-dependent proliferation of T cells. European Journal of Immunology, 40, 1496-1503. doi:10.1002/eji.200939574

[21]   Bernard-Kargar, C., Kassis, N., Berthault, M.F., Pralong, W. and Ktorza, A. (2001) Sialylated form of the neural cell adhesion molecule (NCAM): A new tool for the identification and sorting of beta-cell subpopulations with different functional activity. Diabetes, 50, S125-S130. doi:10.2337/diabetes.50.2007.S125

[22]   Minana, R., Sancho-Tello, M., Climent, E., Segui, J.M., Renau-Piqueras, J. and Guerri, C. (1998) Intracellular location, temporal expression, and polysialylation of neural cell adhesion molecule in astrocytes in primary culture. Glia, 24, 415-427. doi:10.1002/(SICI)1098-1136(199812)24:4<415::AID-GLIA7>3.0.CO;2-A

[23]   Seki, T. (2002) Expression patterns of immature neuronal markers PSA-NCAM, CRMP-4 and NeuroD in the hippocampus of young adult and aged rodents. Journal of Neuroscience Research, 70, 327-334. doi:10.1002/jnr.10387

[24]   Luo, L., Yano, N. and Luo, J.Z. (2006) The molecular mechanism of EGF receptor activation in pancreatic beta-cells by thyrotropin-releasing hormone. American Journal of Physiology, 290, E889-E899.

[25]   Vredenburgh, J.J., Silva, O., Tyer, C., et al. (1996) A comparison of immunohistochemistry, two-color immunofluorescence, and flow cytometry with cell sorting for the detection of micrometastatic breast cancer in the bone marrow. Journal of Hematotherapy, 5, 57-62. doi:10.1089/scd.1.1996.5.57

[26]   Li, M., Inaba, M., Guo, K.Q., Hisha, H., Abraham, N.G. and Ikehara, S. (2007) Treatment of streptozotocin-induced diabetes mellitus in mice by intra-bone marrow bone marrow transplantation plus portal vein injection of beta cells induced from bone marrow cells. International Journal of Hematology, 86, 438-445. doi:10.1007/BF02984002

[27]   Chao, K.C., Chao, K.F., Fu, Y.S. and Liu, S.H. (2008) Islet-like clusters derived from mesenchymal stem cells in Wharton’s Jelly of the human umbilical cord for transplantation to control type 1 diabetes. PloS One, 3, e1451. doi:10.1371/journal.pone.0001451

[28]   Dong, Q.Y., Chen, L., Gao, G.Q., et al. (2008) Allogeneic diabetic mesenchymal stem cells transplantation in streptozotocin-induced diabetic rat. Clinical and Investigative Medicine, 31, E328-E337.

[29]   Gao, X., Song, L., Shen, K., Wang, H., Niu, W. and Qin, X. (2008) Transplantation of bone marrow derived cells promotes pancreatic islet repair in diabetic mice. Biochemical and Biophysical Research Communications, 371, 132-137. doi:10.1016/j.bbrc.2008.04.033

[30]   Cantarelli, E., Melzi, R., Mercalli, A., et al. (2009) Bone marrow as an alternative site for islet transplantation. Blood, 114, 4566-4574. doi:10.1182/blood-2009-03-209973

[31]   Figliuzzi, M., Cornolti, R., Perico, N., et al. (2009) Bone marrow-derived mesenchymal stem cells improve islet graft function in diabetic rats. Transplantation Proceedings, 41, 1797-1800. doi:10.1016/j.transproceed.2008.11.015

[32]   Xie, Q.P., Huang, H., Xu, B., et al. (2009) Human bone marrow mesenchymal stem cells differentiate into insulin-producing cells upon microenvironmental manipulation in vitro. Differentiation: Research in Biological Diversity, 77, 483-491.

[33]   Kang, E.M., Zickler, P.P., Burns, S., et al. (2005) Hematopoietic stem cell transplantation prevents diabetes in NOD mice but does not contribute to significant islet cell regeneration once disease is established. Experimental Hematology, 33, 699-705. doi:10.1016/j.exphem.2005.03.008

[34]   Akashi, T., Shigematsu, H., Hamamoto, Y., et al. (2008) Bone marrow or foetal liver cells fail to induce islet regeneration in diabetic Akita mice. Diabetes/Metabolism Research and Reviews, 24, 585-590. doi:10.1002/dmrr.884

[35]   Bennett, D.L., Bailyes, E.M., Nielsen, E., et al. (1992) Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. The Journal of Biological Chemistry, 267, 15229-15236.

[36]   Smeekens, S.P., Montag, A.G., Thomas, G., et al. (1992) Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3. Proceedings of the National Academy of Sciences of the United States of America, 89, 8822-8826. doi:10.1073/pnas.89.18.8822

[37]   Rhodes, C.J. and Halban, P.A. (1987) Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway. The Journal of Cell Biology, 105, 145-153. doi:10.1083/jcb.105.1.145

[38]   Rice, C.M. and Scolding, N.J. (2008) Autologous bone marrow stem cells—Properties and advantages. Journal of the Neurological Sciences, 265, 59-62. doi:10.1016/j.jns.2007.06.011

[39]   Lee, R.H., Seo, M.J., Reger, R.L., et al. (2006) Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice. Proceedings of the National Academy of Sciences of the United States of America, 103, 17438-17443. doi:10.1073/pnas.0608249103

[40]   Kataoka, K., Medina, R.J., Kageyama, T., et al. (2003) Participation of adult mouse bone marrow cells in reconstitution of skin. The American Journal of Pathology, 163, 1227-1231. doi:10.1016/S0002-9440(10)63482-7

[41]   Abedi, M., Foster, B.M., Wood, K.D., et al. (2007) Haematopoietic stem cells participate in muscle regeneration. British Journal of Haematology, 138, 792-801. doi:10.1111/j.1365-2141.2007.06720.x

[42]   Cantarelli, E., Melzi, R., Mercalli, A., et al. (2009) Bone marrow as an alternative site for islet transplantation. Blood, 114, 4566-4574.

[43]   Hasegawa, Y., Ogihara, T., Yamada, T., et al. (2007) Bone marrow (BM) transplantation promotes beta-cell regeneration after acute injury through BM cell mobilization. Endocrinology, 148, 2006-2015. doi:10.1210/en.2006-1351

[44]   Hess, D., Li, L., Martin, M., et al. (2003) Bone marrow-derived stem cells initiate pancreatic regeneration. Nature Biotechnology, 21, 763-770. doi:10.1038/nbt841

[45]   Johansson, U., Rasmusson, I., Niclou, S.P., et al. (2008) Formation of composite endothelial cell-mesenchymal stem cell islets: A novel approach to promote islet revascularization. Diabetes, 57, 2393-2401. doi:10.2337/db07-0981

[46]   Salazar-Banuelos, A., Wright, J., Sigalet, D. and Benitez-Bribiesca, L. (2008) The bone marrow as a potential receptor site for pancreatic islet grafts. Archives of Medical Research, 39, 139-141. doi:10.1016/j.arcmed.2007.09.004