A novel automated bioreactor for scalable process optimisation of haematopoietic stem cell culture

Proliferation and differentiation of haematopoietic stem cells (HSCs) from umbilical cord blood at large scale will potentially underpin production of a number of therapeutic cellular products in development, including erythrocytes and platelets. However, to achieve production processes that are scalable and optimised for cost and quality, scaled down development platforms that can define process parameter tolerances and consequent manufacturing controls are essential. We have demonstrated the potential of a new, automated, 24×15mL replicate suspension bioreactor system, with online monitoring and control, to develop an HSC proliferation and differentiation process for erythroid committed cells (CD71(+), CD235a(+)). Cell proliferation was relatively robust to cell density and oxygen levels and reached up to 6 population doublings over 10 days. The maximum suspension culture density for a 48h total media exchange protocol was established to be in the order of 10(7)cells/mL. This system will be valuable for the further HSC suspension culture cost reduction and optimisation necessary before the application of conventional stirred tank technology to scaled manufacture of HSC derived products.     

In vivo divisional tracking of hematopoietic stem cells

Hematopoietic stem cell (HSC) division leads to self-renewal, differentiation, or death of HSCs, and adequate balance of this process results in sustained, lifelong, high-throughput hematopoiesis. Despite their contribution to hematopoietic cell production, the majority of cells within the HSC population are quiescent at any given time. Recent studies have tackled the questions of how often HSCs divide, how divisional history relates to repopulating potential, and how many HSCs contribute to hematopoiesis. Here, we summarize these recent findings on HSC turnover from different experimental systems and discuss hypothetical models for HSC cycling and maintenance in steady-state and upon hematopoietic challenge.     

Self-renewal, differentiation or death: regulation and manipulation of hematopoietic stem cell fate.

Hematopoietic stem cells (HSCs) are the rare cells from which all hematopoietic cells are derived. The absence of HSCs is not compatible with life because many essential cells, such as myeloid and erythroid cells, are short lived. The hematopoietic system is the first essential organ system that fails following cytotoxic treatments. It is the vulnerability of HSCs that prevents regeneration following treatment and thus long-term survival. Because HSCs have the capacity to regenerate a functional hematopoietic system, the manipulation of these cells in vitro holds many promises for gene-therapeutic and other applications; however, these are severely curtailed by current difficulties in maintaining and expanding HSCs in culture. This review focuses on recent approaches towards understanding how the HSC compartment is regulated in vivo and discusses how this knowledge might be applied to manipulating HSC numbers.     

Megakaryocytes are essential for HSC quiescence through the production of thrombopoietin

Tissue homeostasis demands regulatory feedback, suggesting that hematopoietic stem cell (HSC) activity is controlled in part by HSC progeny. Yet, cell extrinsic HSC regulation has been well characterized only in niche cells of non-hematopoietic origin. Here we identify feedback regulation of HSCs by megakaryocytes (Mks), which are mature hematopoietic cells, through production of thrombopoietin (Thpo), a cytokine pertinent for HSC maintenance. Induced ablation of Mk cell population in mice perturbed quiescent HSCs in bone marrow (BM). The ablation of Mks resulted in decreased intra-BM Thpo concentration presumably due to Thpo production by Mks. Thpo administration Mk ablated mice restored HSC functions. Overall, our study establishes Mk as an essential cellular component of the HSC niche and delineates cytokine-oriented regulation of HSCs by their own progeny.     

Clinical strategies for expansion of haematopoietic stem cells

Haematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of diseases. The ability to increase the number of HSCs either in vivo or in vitro would provide new treatment options, but the amplification of HSCs has been difficult to achieve. Recent insights into the mechanisms of HSC self-renewal now make the amplification of HSCs a plausible clinical goal. This article reviews the molecular mechanisms that control HSC numbers and discusses how these can be modulated to increase the number of HSCs. Clinical applications of HSC expansion are then discussed for their potential to address the current limitations of HSC transplantation.     

Attenuated response to liver injury in moesin-deficient mice: impaired stellate cell migration and decreased fibrosis

Hepatic stellate cells (HSCs) respond to injury with a coordinated set of events (termed activation), which includes migration and upregulation of matrix protein production. Cell migration requires an intact actin cytoskeleton that is linked to the plasma membrane by ezrin-radixin-moesin (ERM) proteins. We have previously found that the linker protein in HSCs is exclusively moesin. Here, we describe HSC migration and fibrogenesis in moesin-deficient mice. We developed an acute liver injury model that involved focal thermal denaturation and common bile duct ligation. HSC migration and collagen deposition were assessed by immunohistology and quantitative real-time PCR. Activated HSCs were isolated from wild-type or moesin-deficient mice for direct examination of migration. Activated HSCs from wild-type mice were positive for moesin. Migration of moesin-deficient HSCs was significantly reduced. In a culture assay, 22.1% of normal HSCs migrated across a filter in 36h. In contrast, only 1.3% of activated moesin-deficient HSCs migrated. Collagen deposition around the injury area similarly was reduced in moesin-deficient liver. The linker protein moesin is essential for HSC activation and migration in response to injury. Fibrogenesis is coupled to migration and reduced in moesin-deficient mice. Agents that target moesin may be beneficial for chronic progressive fibrosis.     

The role of Wnt signaling in hematopoietic stem cell development

Hematopoietic stem cells (HSCs) can self-renew and differentiate into all cell types of the blood. This is therapeutically important as HSC transplants can provide a curative effect for blood cancers and disorders. The process by which HSCs develop has been the subject of extensive research in a variety of model organisms; however, efforts to produce bonafide HSCs from pluripotent precursors capable of long-term multilineage reconstitution have fallen short. Studies in zebrafish, chicken, and mice have been instrumental in guiding efforts to derive HSCs from human pluripotent stem cells and have identified a complex set of molecular signals and cellular interactions mediated by such developmental regulators as fibroblast growth factor, Notch, transforming growth factor beta (TGFβ), and Wnt, which collectively promote the stepwise developmental progression toward mature HSCs. Tight temporal and spatial control of these signals is critical to generate the appropriate numbers of HSCs needed for the life of the organism. The role of the Wnt family of signaling proteins in hematopoietic development has been the subject of many studies owing in part to the complex nature of its signaling mechanisms. By integrating cell fate specification with cell polarity establishment, Wnt is uniquely capable of controlling complex biological processes, including at multiple stages of embryonic HSC development, from HSC specification to emergence from the hemogenic epithelium to subsequent expansion. This review highlights key signaling events where specific Wnt signals instruct and guide hematopoietic development in both zebrafish and mice and extend these findings to current efforts of generating HSCs in vitro.     

Loss of expression of miR-335 is implicated in hepatic stellate cell migration and activation

Activation and migration of resident stellate cells (HSCs) within the hepatic space of Disse play an important role in hepatic fibrosis, which accounts for the increased numbers of activated HSCs in areas of inflammation during hepatic fibrosis. Currently, microRNAs have been found to play essential roles in HSC differentiation, proliferation, apoptosis, fat accumulation and collagen production. However, little is known about microRNA mediated HSC activation and migration. In this study, the miRNA expression profiles of quiescent HSCs, partially activated HSCs and fully activated HSCs were compared in pairs. Gene ontology (GO) and GO-Map network analysis indicated that the activation of HSCs was regulated by microRNAs. Among them miR-335 was confirmed to be significantly reduced during HSC activation by qRT-PCR, and restoring expression of miR-335 inhibited HSC migration and reduced α-SMA and collagen type I. Previous study revealed that tenascin-C (TNC), an extracellular matrix glycoprotein involved in cell migration, might be a target of miR-335. Therefore, we further studied the TNC expression in miR-335 over-expressed HSCs. Our data showed that exogenous TNC could enhance HSC migration in vitro and miR-335 restoration resulted in a significant inhibition of TNC expression. These results demonstrated that miR-335 restoration inhibited HSC migration, at least in part, via downregulating the TNC expression.     

Hematopoietic stem cells: generation and self-renewal

Adult stem cells hold great promise for future therapeutic applications. Hematopoietic stem cells (HSCs) are among the best-characterized adult stem cells. As such, these cells provide a conceptual framework for the study of adult stem cells from other organs. Here, we review the current knowledge of HSC generation during embryonic development and HSC maintenance in the bone marrow (BM) during adult life. Recent scientific progress has demonstrated that the development of HSCs involves many anatomical sites in the embryo, but the relative contribution of each of these sites to the adult HSC pool remains controversial. Specialized anatomical sites in the BM have been identified as stem cell niches, and these play essential roles in regulating the self-renewal and differentiation of HSCs through recently identified signaling pathways. Extracellular signaling from stem cell niches must integrate with the intracellular molecular machinery and/or genetic programs to regulate HSC fate choice. The exact cellular and/or molecular mechanisms defining stem cell niche and 'stemness' of HSC is largely unknown although substantial progress has been made recently. Hence, many questions remain to be answered even in this relatively well-defined model of stem cell biology.     

Tenascin-C promotes migration of hepatic stellate cells and production of type I collagen

Tenascin-C (TN-C) is an extracellular matrix glycoprotein markedly upregulated during liver fibrosis. The study is performed to explore the role of TN-C during the growth and activation of hepatic stellate cells (HSCs). We found that TN-C was accumulated accompanying with the HSC activation. Our data on cell migration assay revealed that the rTN-C treatment enhanced HSC migration in a dose- and time-dependent manner, but did not influence their proliferation. HSCs transfected with pTARGET-TN-C overexpression vector displayed increased the type I collagen (Col I) production. TN-C overexpression enhanced the process of HSC activation through TGF-β1 signaling. Moreover, the anti-α9β1 integrin antibody treatment blocked the TN-C-driven Col I increase in rat HSCs. Collectively, TN-C had a positive role in activation of HSCs mediated by TGF-β1 and α9β1 integrin, manifesting elevation of Col I production and promotion of cell migration. Our results provide a potential insight for the therapy of hepatic fibrosis.     

Metabolic regulation of hematopoietic stem cells in the hypoxic niche

Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) reside in a hypoxic bone marrow environment, and their metabolic status is distinct from that of their differentiated progeny. HSCs generate energy mainly via anaerobic metabolism by maintaining a high rate of glycolysis. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species, but leaves HSCs susceptible to changes in redox status. In this review, we discuss the importance of oxygen homeostasis and energy metabolism for maintenance of HSC function and long-term self-renewal.     

Total body irradiation causes residual bone marrow injury by induction of persistent oxidative stress in murine hematopoietic stem cells

Ionizing radiation (IR) and/or chemotherapy causes not only acute tissue damage but also late effects including long-term (or residual) bone marrow (BM) injury. The induction of residual BM injury is primarily attributable to the induction of hematopoietic stem cell (HSC) senescence. However, the molecular mechanisms by which IR and/or chemotherapy induces HSC senescence have not been clearly defined, nor has an effective treatment been developed to ameliorate the injury. Thus, we investigated these mechanisms in this study. The results from this study show that exposure of mice to a sublethal dose of total body irradiation (TBI) induced a persistent increase in reactive oxygen species (ROS) production in HSCs only. The induction of chronic oxidative stress in HSCs was associated with sustained increases in oxidative DNA damage, DNA double-strand breaks (DSBs), inhibition of HSC clonogenic function, and induction of HSC senescence but not apoptosis. Treatment of the irradiated mice with N-acetylcysteine after TBI significantly attenuated IR-induced inhibition of HSC clonogenic function and reduction of HSC long-term engraftment after transplantation. The induction of chronic oxidative stress in HSCs by TBI is probably attributable to the up-regulation of NADPH oxidase 4 (NOX4), because irradiated HSCs expressed an increased level of NOX4, and inhibition of NOX activity with diphenylene iodonium but not apocynin significantly reduced TBI-induced increases in ROS production, oxidative DNA damage, and DNA DSBs in HSCs and dramatically improved HSC clonogenic function. These findings provide the foremost direct evidence demonstrating that TBI selectively induces chronic oxidative stress in HSCs at least in part via up-regulation of NOX4, which leads to the induction of HSC senescence and residual BM injury.     

Mn(III) meso-tetrakis-(N-ethylpyridinium-2-yl) porphyrin mitigates total body irradiation-induced long-term bone marrow suppression

Our recent studies showed that total body irradiation (TBI) induces long-term bone marrow (BM) suppression in part by induction of hematopoietic stem cell (HSC) senescence through reactive oxygen species (ROS). In this study, we examined if Mn(III) meso-tetrakis-(N-ethylpyridinium-2-yl) porphyrin (MnTE), a superoxide dismutase mimetic and potent antioxidant, can mitigate TBI-induced long-term BM injury in a mouse model. Our results showed that post-TBI treatment with MnTE significantly inhibited the increases in ROS production and DNA damage in HSCs and the reduction in HSC frequency and clonogenic function induced by TBI. In fact, the clonogenic function of HSCs from irradiated mice after MnTE treatment was comparable to that of HSCs from normal controls on a per-HSC basis, suggesting that MnTE treatment inhibited the induction of HSC senescence by TBI. This suggestion is supported by the finding that MnTE treatment also reduced the expression of p16^Ink4a (p16) mRNA in HSCs induced by TBI and improved the long-term and multilineage engraftment of irradiated HSCs after transplantation. Therefore, the results from this study demonstrate that MnTE has the potential to be used as a therapeutic agent to mitigate TBI-induced long-term BM suppression by inhibiting ionizing radiation-induced HSC senescence through the ROS-p16 pathway.     

The endoplasmic reticulum chaperone protein GRP94 is required for maintaining hematopoietic stem cell interactions with the adult bone marrow niche

Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. GRP94, an endoplasmic reticulum chaperone, has been reported to be essential for the expression of specific integrins and to selectively regulate early T and B lymphopoiesis. In GRP94 deficient BM chimeras, multipotent hematopoietic progenitors persisted and even increased, however, the mechanism is not well understood. Here we employed a conditional knockout (KO) strategy to acutely eliminate GRP94 in the hematopoietic system. We observed an increase in HSCs and granulocyte-monocyte progenitors in the Grp94 KO BM, correlating with an increased number of colony forming units. Cell cycle analysis revealed that a loss of quiescence and an increase in proliferation led to an increase in Grp94 KO HSCs. This expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild-type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT control, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Investigating this, we further determined that there was a near complete loss of integrin α4 expression on the cell surface of Grp94 KO HSCs, which showed impaired binding with fibronectin, an extracellular matrix molecule known to play a role in mediating HSC-niche interactions. Furthermore, the Grp94 KO mice displayed altered myeloid and lymphoid differentiation. Collectively, our studies establish GRP94 as a novel cell intrinsic factor required to maintain the interaction of HSCs with their niche, and thus regulate their physiology.     

On the origin of hematopoietic stem cells: progress and controversy

Hematopoietic Stem Cells (HSCs) are responsible for the production and replenishment of all blood cell types during the entire life of an organism. Generated during embryonic development, HSCs transit through different anatomical niches where they will expand before colonizing in the bone marrow, where they will reside during adult life. Although the existence of HSCs has been known for more than fifty years and despite extensive research performed in different animal models, there is still uncertainty with respect to the precise origins of HSCs. We review the current knowledge on embryonic hematopoiesis and highlight the remaining questions regarding the anatomical and cellular identities of HSC precursors.     

An<Emphasis Type="Italic">in vitro</Emphasis> method to study the effects of hematopoietic regulators during immune and blood cell development

In adults, hematopoiesis occurs in bone marrow (BM) through a complex process with differentiation of hematopoietic stem cells (HSCs) to immune and blood cells. Human HSCs and their progenitors express CD34. Methods on hematopoietic regulation are presented to show the effects of the chemokine, stromal-derived growth factor (SDF)-1α and the neuropeptide, substance P (SP). SDF-1α production in BM stroma causes interactions with HSCs, thereby retaining the HSCs in regions close to the endosteum, at low oxygen. Small changes in SDF-1α levels stimulate HSC functions through direct and indirect mechanisms. The indirect method occurs by SP production, which stimulates CD34⁺ cells, supported by ligand-binding studies, long-term culture-initiating cell assays for HSC functions, and clonogenic assays for myeloid progenitors. These methods can be applied to study other hematopoietic regulators.