The ROCK Inhibitor Y-27632 Negatively Affects the Expansion/survival of Both Fresh and Cryopreserved Cord Blood-derived CD34+ Hematopoietic Progenitor Cells: Y-27632 Negatively Affects the Expansion/survival of CD34+HSPCs

Overview

Journal Stem Cell Rev Rep

Publisher Springer

Specialty Cell Biology

Date 2010 Feb 25

PMID 20180051

Citations 25

Authors

Clara Bueno

Rosa Montes

Pablo Menendez

Affiliations

Soon will be listed here.

Abstract

Cord blood (CB) is an unlimited source of hematopoietic stem and progenitor cells (HSPC). The use of cryopreserved CB-derived CD34+ HSPCs is successful in children and usually leads to rapid hematopoietic recovery upon transplantation. However, current methods for ex vivo expansion of HSPCs still result in a loss of multilineage differentiation potential and current freeze-thawing protocols result in significant cell death and loss of CD34+ HSPCs. The major cause for the loss of viability after slow freezing is apoptosis induced directly by cryoinjury. Very recent reports have demonstrated that Y-27632, a selective and robust ROCK inhibitor is a potent inhibitor of the apoptosis and is efficient in enhancing the post-thaw survival and recovery of different human stem cells including human embryos, hESCs, induced pluripotent stem cells and mesenchymal stem cells. Here, we analyzed the effect of such an inhibitor in CB-derived CD34+ HSPCs. CB-derived CD34+ HSPCs were MACS-isolated and treated with or without 10 microM of Y-27632. The effect of Y-27632 on culture homeostasis was determined in both fresh and cryopreserved CB-derived CD34+ HSPCs. Our results indicate that the Y-27632 not only dramatically inhibits cell expansion of both fresh and cryopreserved CD34+ HSPCs but also impairs survival/recovery of CD34+ HSPCs upon thawing regardless whether Y-27632 is added to both the cryopreservation and the expansion media and or just to the expansion culture medium with or without hematopoietic cytokines. This study identifies for the first time a detrimental effect of Y-27632 on the expansion and survival of both fresh and cryopreserved CB-derived CD34+ HSPCs, suggesting that Y-27632 may have a differential impact on distinct lineage/tissue-specific stem cells. Our data suggest different functions of Y-27632 on human stem cells growing in suspension versus those growing attached to either treated tissue culture plastic or extracellular matrix. We discourage any clinical application of Y-27632 in potential technical developments aimed at improving cryopreservation procedures of CB-derived cells and/or in vitro expansion of HSPCs without spontaneous differentiation.

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References

Li X, Krawetz R, Liu S, Meng G, Rancourt D . ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum Reprod. 2008; 24(3):580-9. DOI: 10.1093/humrep/den404. View

Menendez P, Bueno C, Wang L, Bhatia M . Human embryonic stem cells: potential tool for achieving immunotolerance?. Stem Cell Rev. 2006; 1(2):151-8. DOI: 10.1385/SCR:1:2:151. View

Gauthaman K, Fong C, Bongso A . Effect of ROCK inhibitor Y-27632 on normal and variant human embryonic stem cells (hESCs) in vitro: its benefits in hESC expansion. Stem Cell Rev Rep. 2009; 6(1):86-95. DOI: 10.1007/s12015-009-9107-8. View

Catalina P, Bueno C, Montes R, Nieto A, Ligero G, Sanchez L . Genetic stability of human embryonic stem cells: A first-step toward the development of potential hESC-based systems for modeling childhood leukemia. Leuk Res. 2008; 33(7):980-90. DOI: 10.1016/j.leukres.2008.08.028. View

Menendez P, Wang L, Chadwick K, Li L, Bhatia M . Retroviral transduction of hematopoietic cells differentiated from human embryonic stem cell-derived CD45(neg)PFV hemogenic precursors. Mol Ther. 2004; 10(6):1109-20. DOI: 10.1016/j.ymthe.2004.08.016. View

Hall A . Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Biol. 1994; 10:31-54. DOI: 10.1146/annurev.cb.10.110194.000335. View

Clarke D, Yadock D, Nicoud I, Mathew A, Heimfeld S . Improved post-thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular-like cryopreservation solution. Cytotherapy. 2009; 11(4):472-9. PMC: 3021966. DOI: 10.1080/14653240902887242. View

Martin-Ibanez R, Unger C, Stromberg A, Baker D, Canals J, Hovatta O . Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor. Hum Reprod. 2008; 23(12):2744-54. DOI: 10.1093/humrep/den316. View

Menendez P, Prosper F, Bueno C, Arbona C, San Miguel J, Garcia-Conde J . Sequential analysis of CD34+ and CD34- cell subsets in peripheral blood and leukapheresis products from breast cancer patients mobilized with SCF plus G-CSF and cyclophosphamide. Leukemia. 2001; 15(3):430-9. DOI: 10.1038/sj.leu.2402051. View

10.

Wang L, Menendez P, Cerdan C, Bhatia M . Hematopoietic development from human embryonic stem cell lines. Exp Hematol. 2005; 33(9):987-96. DOI: 10.1016/j.exphem.2005.06.002. View

11.

Bueno C, Montes R, Martin L, Prat I, Hernandez M, Orfao A . NG2 antigen is expressed in CD34+ HPCs and plasmacytoid dendritic cell precursors: is NG2 expression in leukemia dependent on the target cell where leukemogenesis is triggered?. Leukemia. 2008; 22(8):1475-8. DOI: 10.1038/leu.2008.134. View

12.

Catalina P, Montes R, Ligero G, Sanchez L, De La Cueva T, Bueno C . Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties?. Mol Cancer. 2008; 7:76. PMC: 2567976. DOI: 10.1186/1476-4598-7-76. View

13.

Berz D, McCormack E, Winer E, Colvin G, Quesenberry P . Cryopreservation of hematopoietic stem cells. Am J Hematol. 2007; 82(6):463-72. PMC: 2075525. DOI: 10.1002/ajh.20707. View

14.

Menendez P, Perez-Simon J, Mateos M, Caballero M, Gonzalez M, San-Miguel J . Influence of the different CD34+ and CD34- cell subsets infused on clinical outcome after non-myeloablative allogeneic peripheral blood transplantation from human leucocyte antigen-identical sibling donors. Br J Haematol. 2002; 119(1):135-43. DOI: 10.1046/j.1365-2141.2002.03794.x. View

15.

Gluckman E, Rocha V . Cord blood transplantation: state of the art. Haematologica. 2009; 94(4):451-4. PMC: 2663606. DOI: 10.3324/haematol.2009.005694. View

16.

Claassen D, Desler M, Rizzino A . ROCK inhibition enhances the recovery and growth of cryopreserved human embryonic stem cells and human induced pluripotent stem cells. Mol Reprod Dev. 2009; 76(8):722-32. PMC: 3257892. DOI: 10.1002/mrd.21021. View

17.

Koyanagi M, Takahashi J, Arakawa Y, Doi D, Fukuda H, Hayashi H . Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors. J Neurosci Res. 2007; 86(2):270-80. DOI: 10.1002/jnr.21502. View

18.

Pakzad M, Totonchi M, Taei A, Seifinejad A, Hassani S, Baharvand H . Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging. Stem Cell Rev Rep. 2009; 6(1):96-107. DOI: 10.1007/s12015-009-9103-z. View

19.

Bueno C, Catalina P, Melen G, Montes R, Sanchez L, Ligero G . Etoposide induces MLL rearrangements and other chromosomal abnormalities in human embryonic stem cells. Carcinogenesis. 2009; 30(9):1628-37. DOI: 10.1093/carcin/bgp169. View

20.

Heng B . Effect of Rho-associated kinase (ROCK) inhibitor Y-27632 on the post-thaw viability of cryopreserved human bone marrow-derived mesenchymal stem cells. Tissue Cell. 2009; 41(5):376-80. DOI: 10.1016/j.tice.2009.01.004. View