Hematopoietic stromal cells and megakaryocyte development

Abstract

The hematopoietic microenvironment, and in particular the hematopoietic stromal cell element, are intimately involved in megakaryocyte development. The process of megakaryocytopoiesis occurs within a complex bone marrow microenvironment where adhesive interactions, chemokines, as well as cytokines play a pivotal role. Here we review the effect of stromal cells and cytokines on megakaryocytopoiesis with the aim of exploring new therapeutic strategies for platelet recovery after hematopoietic stem cell transplantation (HSCT).

Keywords:

• Hematopoietic microenvironment
• Stromal cells
• Cytokines
• Megakaryocytopoiesis

Introduction

Prolonged thrombocytopenia is a serious complication of high‐dose chemotherapy in the context of autologous or allogeneic transplantation of hematopoietic stem cells (HSCs).1,2 Platelet transfusions are required to support platelet recovery in such patients. In recent years, experiments utilizing ex vivo expansion of megakaryocytes (MKs) from bone marrow (BM), and peripheral blood and cord blood cells have been developed in order to facilitate the platelet recovery.3 However, megakaryocyte development is governed by a complex network of hematopoietic microenvironment, including the hematopoietic stromal cells, cell adhesion molecular, and cytokines. Damage to this microenvironment is one of the primary causes of hematopoietic stem cell transplantation (HSCT) failure; however, stromal cells could be used to restore this microenvironment.4,5 We review the effect of hematopoietic microenvironment on megakaryocytopoiesis, in order to analyse the mechanisms of platelet therapy. We postulate that promoting the development of MKs with stromal cells may be of research value and clinical significance.

Hematopoietic Microenvironment

The hematopoietic microenvironment consists of stromal cells, extracellular matrixes, and cytokines. It is believed that the secretion of various cytokines and growth factors by hematopoietic microenvironment plays an important role in megakaryocytopoiesis6 and in the enhancement of HSCs engraftment in stem cell transplantation.7,8 Megakaryocyte development is coordinated and controlled by combinations of cytokines and mediators presented within specialized bone marrow ‘niches’.9 Two microenvironmental niches are well described: the vascular niche and the osteoblastic niche. The megakaryocytes thrive at vascular niches, whereas the endosteal niche is specifically important in maintaining the stem cell reserve in the quiescent state. Normally, megakaryocytic cytoplasmic processes penetrate the endothelium and release platelets as a shower (thrombopoiesis).10,11

Human bone marrow‐derived stromal cells (hBMSCs)

There is a long‐standing controversy about the origins of stromal cells. On the one side, it is thought that stromal cells are homologous to hematopoietic cells. In the early stage of hematogenesis, ‘common stem cells’ differentiate into stromal cells, forming the hematopoietic microenvironment, before finally differentiating into hematopoietic cells.12^–15 Another group of researchers argues that human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors, and therefore do not originate from hematopoietic cells.16 Bone marrow stromal cells are derived from mesenchymal stem cells (MSCs), including fibroblasts, adipocytes, endothelial cells, and osteoblasts.17,18 It is noteworthy that MSCs, as the precursor cells of hBMSCs, were were proposed a term ‘multipotent mesenchymal stromal cells’ (with the acronym ‘MSCs’) by the International Society for Cellular Therapy.19,20

Stromal cells are firstly and generally found in bone marrow. Majumdar et al.21 reported that primary and passage 1 cultures of hBMSCs contain a heterogeneous population of hematopoietic and stromal cells, After 14 days, primary cultures of hBMSCs show evidence of hematopoiesis, as indicated by formation of cobblestone area formation. Conventional wisdom has held that stromal cells in Dexter cultures comprise a mixture of macrophages, hematopoietic cells, adipocytes, osteoblasts, fibroblasts, muscle cells, and endothelial cells. However, there is evidence proved that the hBMSCs in Dexter cultures actually represent a single cell type (multi‐differentiated mesenchymal progenitor cell).22 Stromal cells are the progeny of mesenchymal cells following a differentiation pathway of vascular smooth muscle cells.23 Stromal cells from all lines, express mesenchymal markers (vimentin, laminin‐beta1, fibronectin, and osteopontin). Phenotypically, ex vivo expanded stromal cells express a number of non‐specific markers, including Stro‐1,24 CD10,25 SH2, SH3, SH4,26 CD90,27 CD105 (endoglin),28 and CD106.29 hBMSCs are devoid of hematopoietic and endothelial markers, such as CD11b, CD14, CD31, and CD45.30

Significant clinical expectations have been associated with three functional aspects of hBMSCs: tissue repair, immune modulation, as well as hematopoietic support.31 hBMSCs show similar cytokine and growth factor expression as MSCs.21 hBMSCs are capable of supporting hematopoiesis by demonstrating the expression of several hematopoietic growth factors and extracellular matrix receptors including thrombopoietin (TPO), interleukins, granulocyte colony‐stimulating factor (G‐CSF), stem cell factor (SCF), vascular cell adhesion molecule‐1 (VCAM‐1), intercellular cell adhesion molecule‐1 (ICAM‐1), activated leukocyte cell adhesion molecule, stromal cell‐derived factor‐1 (SDF‐1), and fibroblast growth factor (FGF), all of which act directly on hematopoietic cells via the paracrine pathway, or combine with extracellular matrix to regulate hematogenesis.32

Human umbilical cord blood‐derived stromal cells (hUCBDSCs)

Human umbilical cord blood is abundant, collected easily, and has a low probability of pathophoresis.33 Therefore, human umbilical cord blood is often used as an alternative and attractive source for cellular or gene therapy.

hUCBDSCs are isolated and identified from cord blood. Following prolonged culture, cells are mainly composed of macrophage‐like, fibroblast‐like, and small‐sphere‐like cells.34 By immunocytochemistry stain, the hUCB‐derived stromal cells are positive for CD106, CD29, CD44, CD45, CD50, CD68, and HLA‐ I, and negative for CD34, HLA‐ II, and T‐cell co‐stimulatory molecule CD80, CD86, CD40 and CD40L.34^–36 hUCB‐derived stromal cells can excrete hematopoietic factors, such as TPO, SDF‐1, GM‐CSF, and SCF. These factors may be closely linked with megakaryocyte development.37,38 Moreover, hUCBDSCs are superior to hBMSCs in promoting CFU‐Meg formation and the recovery of platelets in nude mice after transplant. This suggests that hUCBDSCs may play a unique role in megakaryocytopoiesis.38

Cell adhesion molecules (CAMs)

CAMs play a key role in cell‐cell interactions between hematopoietic stromal cells and MK progenitor cells in bone marrow. CAMs include extracellular matrix molecules, integrins, selectins, cadherin, and immunoglobulin superfamily, such as VCAM‐1, very late antigen (VLA‐4 and ‐5), platelet endothelial cell adhesion molecule‐1(PECAM‐1), and ICAM‐1.

Integrins are reported to be involved in the adhesion of megakaryocytes to fibronectin and fibrinogen, which indicates that integrins have a role in MK maturation and platelet production.39 The VLA‐4 is a receptor for VCAM‐1. VCAM‐1 is expressed by cultured BM stromal cells.40,41 Activation of VLA‐4 and ‐5 can induce the adhesion of megakaryocytic leukemia‐derived cell line Mo7e to fibronectin and VCAM‐1.42 Previous studies have shown that VCAM‐1/VLA‐4 adhesion molecules support the attachment of megakaryocytes to bone marrow endothelial cells, as well as bone marrow fibroblasts.43^–45 It was found that megakaryocytes in PECAM‐1^−/− mice cannot migrate along with the SDF‐1 concentration gradient.46 Subsequent studies reported that PECAM‐1 on the megakaryocytes can chemotactically cause megakaryocytes to migrate by changing the distribution of its own CXCR4.46,47 There is report which suggests that the occurrence of megakaryocytic emperipoiesis is partly dependent on adhesion molecules via LFA‐1/ICAM‐1.48 On the other hand, high levels of ICAM‐1 may be one of the factors initiating the events ultimately leading to clonal thrombocytosis, a pathological unregulated MKs development and platelet production.49 Selectins (CD62L and CD62P) can mediate megakaryocyte–fibroblast interactions in human bone marrow.50 Taken together, CAMs mainly mediate MKs adhesion to hematopoietic microenvironment, so as to regulate the development of MKs.

Cytokines

The regulation of megakaryocyte development appears to be controlled not only by the interactions between stromal cells and MKs, or the cell adhesion molecules and MKs, but also by soluble cytokines and chemokines. During the megakaryocytopoiesis, the nascent stage of MK progenitor cells is regulated primarily by TPO and to a lesser degree by other cytokines, such as IL‐1, IL‐3, and platelet‐derived growth factor (PDGF). Later differentiation stages are regulated primarily again by TPO and probably by IL‐6 and IL‐11.51^–54 In addition, SDF‐1 may play its role in the promotion of megakaryocytopoiesis (Table 1).55,56 Important cell factors include:

Table 1. Cytokines in megakaryocytopoiesis

Major growth factor	TPO (c‐mpl ligand)
Others	IL‐1beta, IL‐3, IL‐6, IL‐7, IL‐9, IL‐11, PDGF, FGF‐2. FGF‐4, Flt‐3 ligand, stem cell factor, GM‐CSF, EPO, SDF‐1

References 27,54,59,62,63,66,67,82,83,86.

1.	TPO: BM stromal cells are known to synthesize TPO.27 TPO is the primary physiological growth factor for the MK lineage, which can stimulate the proliferation and maturation of MK progenitor cells. It stimulates MKs to increase in cell size and ploidy, and to form proplatelet processes that then fragment into single platelets.57–59 Furthermore, when purified MKs were co‐cultured with stromal cells, most of the MKs adhered to the stromal cells and remained unchanged, while free MKs induced proplatelet formation. Thus, the interaction of MKs with stromal cells may suppress platelet formation.60 Nagahisa et al.61 believed that hBMSCs produce TPO and stimulate MK growth and maturation, but suppress the formation of lengthy beaded cytoplasmic processes;
2.	SDF‐1: SDF‐1 is a chemotactic factor produced by stromal cells. SDF‐1 supports megakaryocytopoiesis and homing of HSCs to the BM during fetal development.62 SDF‐1, together with FGF‐4, supports platelet production in TPO−/− or mpl−/− mice through interactions of MK progenitors with the BM vascular niche.59,63 The activity of SDF‐1 is important for the movement of megakaryocyte progenitors from the proliferative ‘osteoblastic niche’ to the ‘vascular niche’ for platelet formation.63,64 Additionally, platelet production is enhanced during transendothelial migration of CXCR4+ MK in response to SDF‐1.65 Perez et al.55 have found that SDF‐1 and its analogue may be of clinical value in stimulating platelet recovery after chemo/radiation treatment as well as in stem cell mobilization;
3.	IL: the stromal cells can express mRNAs of IL‐1 through IL‐7 in bone marrow and fetal liver stromal cells.66 IL‐1beta, IL‐3, and IL‐6, together with PDGF, are reported to increase the megakaryocytic progenitors (CD61+CD41+ cells and CFU‐MK).67–69 IL‐6 and IL‐7 both derive from stromal cells and exert important effects in terms of MKs proliferation and differentiation.70–72 Fujiki et al.73 demonstrated that human IL‐9 can potentiate human megakaryocytopoiesis in the presence of erythropoietin (EPO) and/or SCF. IL‐11 is isolated from a marrow stromal fibroblast cell line, and this cytokine plays a key role in the regulation of human megakaryocytopoiesis in the BM. In the presence of rhIL‐3, rhIL‐11 stimulated an increase in the number, size, and ploidy value of the megakaryocyte colonies by non‐adherent, T cell‐depleted marrow mononuclear cells;74,75
4.	SCF: it is suggested that a close cell‐to‐cell interaction between MKs and stromal cells is important for an optimal stimulation of megakaryocytopoiesis through the SCF/c‐kit receptor system.76 SCF is a ligand of the protein tyrosine kinase receptor c‐kit, which is found in two forms: transmembrane SCF (tm‐SCF) and soluble SCF (s‐SCF). Research in vitro indicates that tm‐SCF can induce CD34+ cell proliferation over time, but soluble SCF cannot.77 SCF can increase the number of megakaryocytes per colony in the presence of IL‐3, GM‐CSF, or IL‐6. SCF also stimulates the proliferation of specific megakaryocytic cell lines (CMK and M‐07e).78 SCF is recognized as a hematopoietic growth factor, acting on nascent hematopoietic stem cells or progenitor cells;
5.	FL: just like the SCF, FL is also produced by BM stromal cells. It is a ligand of protein tyrosine kinase receptors, and includes two types: soluble and combined. The soluble form is known to be active. FL synergistically augments the ability of IL‐3 and c‐kit ligand, alone or in association, to promote long‐term megakaryocytopoiesis.79 Flt3/Flk‐2 ligand in combination with TPO increases the numbers of megakaryocyte progenitor cells in serum‐free cultures, and decreases apoptosis in megakaryocyte development;80,81
6.	FGF: expression of FGFs can be found in various tissue and cells, including BM stromal cells, smooth muscle cells, myocardial cells, etc. FGF as a potent mitogen, especially FGF‐282 and FGF‐4,83,84 has been reported to improve the performance of human long‐term hematopoietic cultures. Basic FGF can enhance the adhesion of BM megakaryocytes to marrow stromal fibrobasts and induce cytokine secretion (IL‐6, IL‐1beta, and GM‐CSF).43 Furthermore, it is suggested that FGF is capable of stimulating megakaryocytopoiesis and the effect of FGF is mediated through an interaction with FGF receptor types 1 and 2 located on the megakaryocytic lineage.43,85

Additionally, a number of transcription factors have been implicated in MK differentiation. GATA‐1, FOG‐1, and NF‐E2 are essential regulators of megakaryocytopoiesis. Fli‐1 likely also plays an important role in the process.54,86 In vitro, some chemotactic factors can inhibit megakaryocytopoiesis, such as those in CXC categories (PF4, betaTG, NAP‐2, and IL‐8) and in CC categories (MIP‐1alpha, MIP‐1beta, and IP10).87 Transforming growth factor‐beta1 and interferons, produced by stromal cells, can also inhibit megakaryocyte colony formation88,89 (Table 2).

Table 2. Transcriptional regulation of megakaryocytopoiesis

Megakaryocyte differentiation	Transcription factors	Target genes
Early‐ and mid‐stage	GATA‐1	p45 NF‐E2 Inositol polyphos 4‐P'tase Type I
	FOG‐1	NF‐E2
	CBFA2
Late‐stage and platelet release	NF‐E2	Beta1‐tubulin

References 54,86.

As mentioned above, stromal cells produce essential hematopoietic growth factors, express adhesion molecules and extracellular matrix proteins that are known to play a role in hematopoietic stem cell mobilization and homing, support the growth of hematopoietic stem and progenitor cells, and dramatically enhance megakaryocyte and platelet formation in vitro.7,90 This evidence suggests that co‐transplantation of stromal cells could facilitate HSC engraftment in patients. Indeed, several studies have already reported that co‐transplantation of stromal cells can enhance the success of HSCT,91 prevent graft‐versus‐host disease,92 and promote myelopoiesis and megakaryocytopoiesis.7

Conclusions and Perspectives

In conclusion, the accumulation of knowledge about molecular mechanisms regulating megakaryocytopoiesis and platelet production provides a strong theoretical basis for the therapy of thrombocytopenic states. The hematopoietic microenvironment, in particular the hematopoietic stromal cells in it, is regarded as the ‘soil’ of HSCs, and shed a light on the physician. For example, ex vivo expansion of MKs through co‐culturing with the stromal cells or through an optimal cytokine combination and in vivo transfusion of hematopoietic stromal cells along with HSCT, might be used as supplemental approaches to conventional grafts to alleviate post‐transplant and chemotherapy‐induced thrombocytopenia.

This study was supported by grants from the National Natural Science Foundation of China (nos. 30800496 and 30971109), and the Chongqing Natural Science Foundation (nos. 2008BB5293 and CSTC2009BA5011).