Cultures of platelet-forming cells (megakaryocytes). Puzzling and dramatically important for regenerative medicine.
Thrombocytes are small nucleated cells involved in preventing blood leak trough capillary injuries (hemostasis) and an important cellular component initiating the wound healing process.
In mammals thrombocyte's functions are assumed by the platelets, which are anucleated cells, actually fragments of cell cytoplasm released from the megakaryocytes, a kind of bone marrow oversized cells that derive from the common hematopoietic stem cells (HSCs). Each platelet receives from the megakaryocyte an active membrane, a quantum of tubules, forming a circular bundle, a quantum of mitochondria, a quantum of glycogen and a package of cytosol molecules needed for platelet activation, clot forming and clot retraction (1). Megakaryocytes are responsible for the continuous production and regeneration of platelet pool in the blood, critical to prevent bleeding.
In living humans the HSCs commit at least to two branches: Myeloid and Lymphoid progenitors. Each progenitor cell further differentiates in more branches with specific commitments. It is known that the lymphoid progenitors yield both B and T lymphocyte precursors, which will differentiate under environmental influence (thymus or bone marrow). All these branches, that are capable of growing and differentiating (maturing) in parallel, will populate the tissues with mature cells by first committing to progenitor cells/colony forming cells (CFU), multiplying in several doubling events and acquiring the mature traits. Myeloid progenitors colony forming units ( CFU-GEMM) sub-commit at list into Granulocyte-macrophage progenitors (CFU-GM), erythroblast progenitors (CFU-e) and megakaryocyt progenitors (CFU-M), but there are data suggesting that under specific signals can also directly sub-commit to Mast cell differentiation (2) and microglia differentiation (3).
Classically it was accepted that the pool of HSCs are specific subtypes biased towards the generation of either lymphoid or myeloid blood cells. However, recently the group of Dr. S.E. Jacobsen (Oxford, England) has published very interesting discoveries: In mouse is possible to identify and isolate a distinct HSC subset primed for platelet-specific gene expression, which is thrombopoietin dependent. Thrombopoietin(THPO) is themegakaryocyte growth and development factor(MGDF) (4). These platelet primed cells can self-renew, maintain for a long time the ability to behave as broad myeloid stem cells and can even give rise to lymphoid-biased progenitors. These discoveries allowed them to suggest that “HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs located at the apex” (5).
If the Megakaryocyt biased HSC is at the highest point of the biased hematopoietic cell hierarchy this is obviously a notable particularity of the Megakaryocytic linage, but not the only one. In general, each CFU derived from the HSCs undergoes between 3 and 6 successive DNA replications and divisions (complete mitosis) generating 8 or up to 64 mature diploid (2n) free cells, except for the megakaryocyt colony forming unit. This cells have two different ways to grow: Either doubling the DNA content and dividing in two diploid cells or doubling the DNA but not followed by cell division (endomitosis) generating polyploid oversized cells. Actually these polyploid cells are comparable to any hematopoietic cell colony because they reach levels of DNA content of 4 to 32 times a diploid nucleus (8n to 64n) (6). Therefore a 64-ployd megakaryocyte is equivalent to 32 diploid cells together in a single body. This colony formation peculiarity of the BFU-MK linage is one of the obstacles to produce megakaryocytes/platelets in culture. Megakaryocytes linage alternate growth and maturation responding to environmental signals not yet fully understood (7) (8). Each megakaryoblast may progress from 2n up to 64n directly with very low level of maturation, until the polyploid process is finished and then it will go through all maturation stages, or it can also reach any intermediate polyploidy and maturate (9). Detailed studies have demonstrated that this last option is the most frequent, that is why in a normal bone marrow megakaryocytes are at any maturation stage of any polyploidy(10). However there are clear evidences that polyploidization and maturation are two processes with separate regulation systems (11)
Schematically, progression of nuclei growth is followed by cytoplasm growth and maturation which occurs in three well defined stages. The lowest maturation stage (s1) is characterized by a slightly granular and intense basophilic cytoplasm of moderate volume surrounding a nucleus of any possible polyploidy. ( 8n, 16n, 32n and 64n). These cells are capable of DNA replication and are responsive to specific cytokines and growth factors but barely initiate the maturation process which basically involves synthesis, packaging and storage of all components of the future platelets.
The intermediate maturation stage (s2) is characterized by a notable granular and pale basophilic cytoplasm of large volume surrounding a nucleus also of any possible polyploidy (8n, 16n, 32n and 64n).At this stage certain platelet demarcations are already visible in limited spots. It is not known for sure that these cells are capable of DNA replication but are responsive to specific cytokines and growth factors showing an advanced level of zonal maturation, which basically involves extended synthesis and storage of all components of the finished platelets and initiation of platelet demarcations in the cytoplasm.
The most advanced maturation stage (s3) is characterized by a large volume acidophilic cytoplasm with intense granulation where the platelet demarcation and even extensions of platelet release are evident. This cytoplasm in disintegration is also surrounding a nucleus of any possible polyploidy (8n, 16n, 32n and 64n). Only a slight minority of these cells may be capable of DNA replication, but they show an advanced level of cytoplasm global maturation, which involves sustained synthesis and storage of all components of the complete platelets and extended formation of platelet demarcations in the cytoplasm, with platelet release dendritic formations (12)
Results obtained in our laboratories (Celartia Research Laboratories –CRL-) demonstrate that the distinction of three additional sorts of megakaryocytes based on the Histones/DNA ratio (H/DNA ratio) of their chromatin can be made.. One kind shows a H/DNA ratio compatible with pre-DNA replication state (13), a second kind have a H/DNA ratio characteristic of post-mitotic state and a third kind has a H/DNA ratio that suggest chromatin condensation and nuclear tendency towards pyknosis (14) (15)
The existence of these sub-kinds of megakaryocytes suggests that in each level of maturation the ability of DNA replication persist, at the same time that the maturation process is advancing, which means that some megakaryocytes can increase the polyploidy in parallel to the maturation process in response to thrombopoietin (TPO) (16).
Therefore, as reported by others, there is no specific level of polyploidy associated to platelet production. However, the existing data suggests that polyploidization ability is canceled when the platelet formation reaches a certain threshold (17).
The analysis of each polyploidy level including all maturation stages showed that megakaryocytes 8n maintain 33% probability of increasing the DNA content. Related to the maturation level, 54.5% of S1 cells may be capable of doubling the polyploidy, however only 25 % of the S2 can. Approximately same pocentage of S3 cells may go into the pyknosis process.
After complete platelet release the nucleus appears necked or coated with a very thin layer of cytoplasm. It has not been completely discarded yet that nuclei with minimal residual coat of cytoplasm will be destroyed or if it could regenerate to a complete megakaryocyte.
All existing research data suggest that Megakaryocytes can be differentiated in cultures of hematopoietic CD34+ stem cells. By adding thrombopoietin (TPO) to the culture media (18) increases megakaryocytes polyploidy or MK-CFU colony expansion in response to the presence of TPO. On the other hand maturation and platelet production are under the influence of other factors, such as vascular endothelial growth factors (VEGFs) and others (19)
Megakaryocytes repopulation and platelet production are two dramatically important goals in hematopoietic regenerative medicine. Many diseases and anti-cancer treatments evolve with dangerous thrombocytopenia (low counts of blood platelets).Although Megakaryocyte transplants from adult bone marrow have demonstrated effective reestablishing platelet counts, transplants of umbilical cord stem cells could be quite ineffective, apparently due to an inefficient adaptation of the umbilical cord stem cells to the bone marrow microenvironment (20).
The prospect of developing productive megakaryocytes cultures is an attractive and logic goal, either culturing megakaryocytes to be transplanted or develop competent platelets in vitro to be injected in the blood stream of emergency treatments.Recent studies showed the feasibility of obtaining CD34+ and CD41+ cells differentiated from hESCs in non immunogenic serum-free, feeder cell- free culture, capable of generating clonogenic Mk progenitors in response to thrombopoietic combinations of cytokines (TPO+GM-CSF+SCF+IL-3+IL-6). These opens up the possibility for functional megakaryocyte generation, from human embryonic stem cells differentiated in vitro to MK-CFU, or even the possible in vitro generation of human platelets under defined conditions for therapeutic use (21). Likewise, it has been confirmed that special cell culture systems may allow in vitro generation of platelets from iPSC-derived megakaryocytes in sufficient amounts for transfusion therapy (22).
REFERENCES
1. Johnson SA. The Circulating Platelet. Elsevier; 2012.
2. Gurish MF, Boyce JA. Mast cell growth, differentiation, and death. Clin Rev Allergy Immunol. 2002 Apr;22(2):107–18.
3. Ajami B, Bennett JL, Krieger C, McNagny KM, Rossi FMV. Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nat Neurosci. 2011 Jul 31;14(9):1142–9.
4. Deutsch VR, Tomer A. Advances in megakaryocytopoiesis and thrombopoiesis: from bench to bedside. Br J Haematol. 2013 Jun;161(6):778–93.
5. Sanjuan-Pla A, Macaulay IC, Jensen CT, Woll PS, Luis TC, Mead A, et al. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature. 2013 Aug 11;
6. Odell TT Jr, Jackson CW. Polyploidy and maturation of rat megakaryocytes. Blood. 1968 Jul;32(1):102–10.
7. Chen S, Su Y, Wang J. ROS-mediated platelet generation: a microenvironment-dependent manner for megakaryocyte proliferation, differentiation, and maturation. Cell Death Dis. 2013;4:e722.
8. D’Atri LP, Pozner RG, Nahmod KA, Landoni VI, Isturiz M, Negrotto S, et al. Paracrine regulation of megakaryo/thrombopoiesis by macrophages. Exp Hematol. 2011 Jul;39(7):763–72.
9. Ebbe S, Carpenter D, Yee T. Megakaryocytopenia in W/Wv mice is accompanied by an increase in size within ploidy groups and acceleration of maturation. Blood. 1989 Jul;74(1):94–8.
10. Paulus JM, Carsten AL, Cronkite EP, Lacaze N. Megakaryocyte Growth in In Vitro Cultures and in Diffusion Chambers. In: MD EPC, Carsten AL, editors. Diffus Chamb Cult [Internet]. Springer Berlin Heidelberg; 1980 [cited 2013 Sep 9]. p. 57–61. Available from: http://link.springer.com/chapter/10.1007/978-3-642-67644-4_6
11. Kikuchi J, Furukawa Y, Iwase S, Terui Y, Nakamura M, Kitagawa S, et al. Polyploidization and functional maturation are two distinct processes during megakaryocytic differentiation: involvement of cyclin-dependent kinase inhibitor p21 in polyploidization. Blood. 1997 Jun 1;89(11):3980–90.
12. Paulus J-M. DNA Metabolism and Development of Organelles in Guinea-Pig Megakaryocytes: A Combined Ultrastructural, Autoradiographic and Cytophotometric Study. Blood. 1970 Mar 1;35(3):298–311.
13. Stone GE. Change in histone-DNA ratio during population growth in Tetrahymena. J Cell Biol. 1969 Sep;42(3):837–40.
14. Burgoyne LA. The mechanisms of pyknosis: hypercondensation and death. Exp Cell Res. 1999 Apr 10;248(1):214–22.
15. Shabalkin IP. [Use of the magnitude of the histone-DNA ratio as a criterion for evaluating the functional activity of the cell genome]. Tsitologiia. 1998;40(1):106–15.
16. Paulus J-M, Debili N, Larbret F, Levin J, Vainchenker W. Thrombopoietin responsiveness reflects the number of doublings undergone by megakaryocyte progenitors. Blood. 2004 Oct 15;104(8):2291–8.
17. Paulus JM. [Ultrastructural and microphotometric study of the maturation of megakaryocytes]. Nouv Rev Française Hématologie. 1968 Jun;8(3):394–7.
18. Siddiqui NFA, Shabrani NC, Kale VP, Limaye LS. Enhanced generation of megakaryocytes from umbilical cord blood-derived CD34(+) cells expanded in the presence of two nutraceuticals, docosahexanoic acid and arachidonic acid, as supplements to the cytokine-containing medium. Cytotherapy. 2011 Jan;13(1):114–28.
19. Thiele W, Krishnan J, Rothley M, Weih D, Plaumann D, Kuch V, et al. VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis. Blood. 2012 Aug 30;120(9):1899–907.
20. Slayton WB, Wainman DA, Li XM, Hu Z, Jotwani A, Cogle CR, et al. Developmental differences in megakaryocyte maturation are determined by the microenvironment. Stem Cells Dayt Ohio. 2005 Oct;23(9):1400–8.
21. Pick M, Azzola L, Osborne E, Stanley EG, Elefanty AG. Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium. PloS One. 2013;8(2):e55530.
22. Nakagawa Y, Nakamura S, Nakajima M, Endo H, Dohda T, Takayama N, et al. Two differential flows in a bioreactor promoted platelet generation from human pluripotent stem cell-derived megakaryocytes. Exp Hematol. 2013 Aug;41(8):742–8.
2. Gurish MF, Boyce JA. Mast cell growth, differentiation, and death. Clin Rev Allergy Immunol. 2002 Apr;22(2):107–18.
3. Ajami B, Bennett JL, Krieger C, McNagny KM, Rossi FMV. Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nat Neurosci. 2011 Jul 31;14(9):1142–9.
4. Deutsch VR, Tomer A. Advances in megakaryocytopoiesis and thrombopoiesis: from bench to bedside. Br J Haematol. 2013 Jun;161(6):778–93.
5. Sanjuan-Pla A, Macaulay IC, Jensen CT, Woll PS, Luis TC, Mead A, et al. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature. 2013 Aug 11;
6. Odell TT Jr, Jackson CW. Polyploidy and maturation of rat megakaryocytes. Blood. 1968 Jul;32(1):102–10.
7. Chen S, Su Y, Wang J. ROS-mediated platelet generation: a microenvironment-dependent manner for megakaryocyte proliferation, differentiation, and maturation. Cell Death Dis. 2013;4:e722.
8. D’Atri LP, Pozner RG, Nahmod KA, Landoni VI, Isturiz M, Negrotto S, et al. Paracrine regulation of megakaryo/thrombopoiesis by macrophages. Exp Hematol. 2011 Jul;39(7):763–72.
9. Ebbe S, Carpenter D, Yee T. Megakaryocytopenia in W/Wv mice is accompanied by an increase in size within ploidy groups and acceleration of maturation. Blood. 1989 Jul;74(1):94–8.
10. Paulus JM, Carsten AL, Cronkite EP, Lacaze N. Megakaryocyte Growth in In Vitro Cultures and in Diffusion Chambers. In: MD EPC, Carsten AL, editors. Diffus Chamb Cult [Internet]. Springer Berlin Heidelberg; 1980 [cited 2013 Sep 9]. p. 57–61. Available from: http://link.springer.com/chapter/10.1007/978-3-642-67644-4_6
11. Kikuchi J, Furukawa Y, Iwase S, Terui Y, Nakamura M, Kitagawa S, et al. Polyploidization and functional maturation are two distinct processes during megakaryocytic differentiation: involvement of cyclin-dependent kinase inhibitor p21 in polyploidization. Blood. 1997 Jun 1;89(11):3980–90.
12. Paulus J-M. DNA Metabolism and Development of Organelles in Guinea-Pig Megakaryocytes: A Combined Ultrastructural, Autoradiographic and Cytophotometric Study. Blood. 1970 Mar 1;35(3):298–311.
13. Stone GE. Change in histone-DNA ratio during population growth in Tetrahymena. J Cell Biol. 1969 Sep;42(3):837–40.
14. Burgoyne LA. The mechanisms of pyknosis: hypercondensation and death. Exp Cell Res. 1999 Apr 10;248(1):214–22.
15. Shabalkin IP. [Use of the magnitude of the histone-DNA ratio as a criterion for evaluating the functional activity of the cell genome]. Tsitologiia. 1998;40(1):106–15.
16. Paulus J-M, Debili N, Larbret F, Levin J, Vainchenker W. Thrombopoietin responsiveness reflects the number of doublings undergone by megakaryocyte progenitors. Blood. 2004 Oct 15;104(8):2291–8.
17. Paulus JM. [Ultrastructural and microphotometric study of the maturation of megakaryocytes]. Nouv Rev Française Hématologie. 1968 Jun;8(3):394–7.
18. Siddiqui NFA, Shabrani NC, Kale VP, Limaye LS. Enhanced generation of megakaryocytes from umbilical cord blood-derived CD34(+) cells expanded in the presence of two nutraceuticals, docosahexanoic acid and arachidonic acid, as supplements to the cytokine-containing medium. Cytotherapy. 2011 Jan;13(1):114–28.
19. Thiele W, Krishnan J, Rothley M, Weih D, Plaumann D, Kuch V, et al. VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis. Blood. 2012 Aug 30;120(9):1899–907.
20. Slayton WB, Wainman DA, Li XM, Hu Z, Jotwani A, Cogle CR, et al. Developmental differences in megakaryocyte maturation are determined by the microenvironment. Stem Cells Dayt Ohio. 2005 Oct;23(9):1400–8.
21. Pick M, Azzola L, Osborne E, Stanley EG, Elefanty AG. Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium. PloS One. 2013;8(2):e55530.
22. Nakagawa Y, Nakamura S, Nakajima M, Endo H, Dohda T, Takayama N, et al. Two differential flows in a bioreactor promoted platelet generation from human pluripotent stem cell-derived megakaryocytes. Exp Hematol. 2013 Aug;41(8):742–8.
