PLoS One. 2015 Aug 27;10(8):e0136652.
Three-Dimensional Environment Sustains Hematopoietic Stem Cell Differentiation into Platelet-Producing Megakaryocytes.
Audrey Pietrzyk-Nivau1, Sonia Poirault-Chassac1, Sophie Gandrille1,2, Sidi-Mohammed Derkaoui3, Alexandre Kauskot1, Didier Letourneur3, Catherine Le Visage3 and Dominique Baruch1
1INSERM, UMR-S 1140, University Paris Descartes, Sorbonne Paris Cité, Paris, France
2AP-HP, Georges Pompidou European Hospital, Department of Hematology, Paris, France
3INSERM, UMR-S 1148, University Paris Diderot, Paris; University Paris Nord, Villetaneuse, Sorbonne Paris Cité, France
Hematopoietic stem cells (HSC) differentiate into megakaryocytes (MK), whose function is to release platelets. Attempts to improve in vitro platelet production have been hampered by the low amplification of MK. Providing HSC with an optimal three-dimensional (3D) architecture may favor MK differentiation by mimicking some crucial functions of the bone marrow structure. To this aim, porous hydrogel scaffolds were used to study MK differentiation from HSC as well as platelet production. Flow cytometry, qPCR and perfusion studies showed that 3D was suitable for longer kinetics of CD34+ cell proliferation and for delayed megakaryocytic differentiation far beyond the limited shelf-life observed in liquid culture but also increased production of functional platelets. We provide evidence that these 3D effects were related to 1) persistence of MK progenitors and precursors and 2) prolongation of expression of EKLF and c-myb transcription factors involved in early MK differentiation. In addition, presence of abundant mature MK with increased ploidy and impressive cytoskeleton elongations was in line with expression of NF-E2 transcription factor involved in late MK differentiation. Platelets produced in flow conditions were functional as shown by integrin αIIbβ3 activation following addition of exogenous agonists. This study demonstrates that spatial organization and biological cues synergize to improve MK differentiation and platelet production. Thus, 3D environment constitutes a powerful tool for unraveling the physiological mechanisms of megakaryopoiesis and thrombopoiesis in the bone marrow environment, potentially leading to an improved amplification of MK and platelet production.
Hematopoietic stem cells (HSC) isolated from hematopoietic tissues are self-renewing, multipotent progenitors of hematopoietic lineages leading to all mature blood cells. In order to improve in vitro HSC proliferation, 3D structures have been used as physical support to the extracellular matrix ECM proteins.
Platelets, which are essential for bleeding arrest, are formed from the enlarged cytoplasm of mature megakaryocytes (MK) (1). Platelet production in vitro remains a challenging task, requiring synergy between biochemical and biophysical factors to reach high platelet yields. Thus, in the aim to improve in vitro platelet production, several novel approaches are currently developed. Hemodynamic shear forces contribute to proplatelet and platelet formation from mature MK in vivo (2) and in vitro (3). A synergy between environment factors has been shown to be involved in i) MK differentiation within the osteoblastic niche of the bone marrow (4), ii) MK migration to the vascular niche (5), and iii) platelet production from fully mature MK (6). 3D biomaterials formed by assembling polymers or biomolecules such as proteins or natural polysaccharides are extensively used in regenerative medicine. Hydrogels have proved valuable for expanding endothelial progenitor cells and for differentiating embryonic stem cells (7, 8). So far, high yields of platelets have been achieved ex vivo from HSC using 3D scaffolds of nonwoven polyester fabric and inverted colloidal crystals/polyacrylamide porous hydrogels, but these platelets seemed activated (9). Nevertheless, MK differentiation leading to this increased platelet production was not investigated in any of these 3D structures.
In this study, we examined how a porous 3D structure affects HSC proliferation, MK differentiation and platelet production. We used 3D scaffolds prepared from polysaccharide (pullulan and dextran)-based hydrogels (10, 11) to reproduce the 3D environment required for CD34+ cell culture in comparison to liquid cell culture.
We first observed cell behavior inside 3D scaffolds compared to liquid culture and we noted that cells in 3D survived for at least 36 days (Figure 1A), whereas cells in liquid culture survived for no more than 16 days (Figure 1B).
Figure 1. Cell proliferation and differentiation in 3D (A) and in liquid culture (B).
Next, we analyzed in vitro megakaryopoiesis inside 3D structures by studying the kinetics of CD34, CD41 and CD42b expression. From day 6 to day 15, CD41 and CD42b expression was similar in 3D and liquid culture. From day 16 to day 36, non-megakaryocytic cells (CD41–/CD42b–; Figure 2A), MK precursors (CD41+/CD42b–; Figure 2B) and mature MK (CD41+/CD42b+; Figure 2C) were still observed in 3D. Moreover, CD34+ cells remained in 3D while they disappeared in liquid culture (Figure 2D). Thus, 3D environment provided a suitable environment for maintenance of hematopoietic progenitor CD34+ cells.
Figure 2. (A, B, C) Frequency of non-megakaryocytic cells (CD41–/CD42b–), MK precursors (CD41+/CD42b–) and mature MK (CD41+/CD42b+) in 3D (closed circles, dotted lines) and liquid culture (open squares, full lines) between day 6 and day 36. Means ±SEM. *p<0.05. (D) CD41/CD34 dot plots of one representative experiment in 3D and liquid culture on days 9, 16 and 23.
Finally, we studied platelet production in flow conditions after recovery of mature and viable MK from 3D compared to liquid culture on day 16. More and larger cytoskeletal elongations were observed from 3D than liquid-culture mature MK (Figure 3A). This increased ability to elongate resulted in a significantly higher percentage of proplatelets/platelets produced by 3D mature MK than liquid-culture MK (Figure 3B). These results demonstrated that this 3D environment allowed the recovery of many platelets.
Figure 3. (A) Stages of MK deformations and reorganization into proplatelets and platelets in 3D (left panel) and in liquid culture (right panel). (B) Histogram representation of proplatelet/platelet numbers as a percentage of total adherent cells per field recovered from mature MK obtained in 3D (black bars) and liquid culture (white bars). Means ±SEM. *p<0.05.
The importance of this study is two-fold. On one hand, 3D environment delayed MK progenitors (CD34+/CD41–) differentiation into MK precursors (CD34–/CD41+) leading to mature MK (CD41+/CD42b+). On the other hand, 3D environment improved enlargement of these mature MK allowing increased platelet production in flow conditions.
(1) Machlus KR, Italiano JE, Jr. (2013) The incredible journey: From megakaryocyte development to platelet formation. J Cell Biol 201(6): 785-796.
(2) Junt T, Schulze H, Chen Z, Massberg S, Goerge T, et al. (2007) Dynamic visualization of thrombopoiesis within bone marrow. Science 317(5845): 1767-1770.
(3) Thon JN, Mazutis L, Wu S, Sylman JL, Ehrlicher A, et al. (2014) Platelet bioreactor-on-a-chip. Blood 124: 1857-1867.
(4) Pallotta I, Lovett M, Rice W, Kaplan DL, Balduini A (2009) Bone marrow osteoblastic niche: a new model to study physiological regulation of megakaryopoiesis. PLoS One 4(12): e8359.
(5) Pallotta I, Lovett M, Kaplan DL, Balduini A (2011) Three-dimensional system for the in vitro study of megakaryocytes and functional platelet production using silk-based vascular tubes. Tissue Eng Part C Methods 17(12): 1223-1232.
(6) Dunois-Larde C, Capron C, Fichelson S, Bauer T, Cramer-Borde E, et al. (2009) Exposure of human megakaryocytes to high shear rates accelerates platelet production. Blood 114(9): 1875-1883.
(7) Lavergne M, Derkaoui M, Delmau C, Letourneur D, Uzan G, et al. (2012) Porous polysaccharide-based scaffolds for human endothelial progenitor cells. Macromol Biosci 12(7): 901-910.
(8) Hamidi S, Letourneur D, Aid-Launais R, Di Stefano A, Vainchenker W, et al. (2014) Fucoidan Promotes Early Step of Cardiac Differentiation from Human Embryonic Stem Cells and Long-Term Maintenance of Beating Areas. Tissue Eng Part A. 20(7-8): 1285-1294.
(9) Sullenbarger B, Bahng JH, Gruner R, Kotov N, Lasky LC (2009) Prolonged continuous in vitro human platelet production using three-dimensional scaffolds. Exp Hematol 37(1): 101-110.
(10) Autissier A, Le Visage C, Pouzet C, Chaubet F, Letourneur D (2010) Fabrication of porous polysaccharide-based scaffolds using a combined freeze-drying/cross-linking process. Acta Biomater 6(9): 3640-3648.
(11) Le Visage C, Gournay O, Benguirat N, Hamidi S, Chaussumier L, et al. (2012) Mesenchymal stem cell delivery into rat infarcted myocardium using a porous polysaccharide-based scaffold: a quantitative comparison with endocardial injection. Tissue Eng Part A 18(1-2): 35-44.
This study was made available through the funding from DIM Stem Pole, INSERM, Universities Paris Descartes, Paris Diderot, Paris Nord and grants from ANR (ANR-09-EBIO-001-01; ANR-09-EBIO-001-09; ANR-10-EMMA-009-01; ANR-11-BSV-010-01).
Dr. Dominique Baruch
INSERM UMR-S 1140
4 avenue de l’Observatoire, 75006 Paris, France