Culturing patient-derived malignant hematopoietic stem cells in engineered and fully humanized 3D niches

Human malignant hematopoietic stem and progenitor cells (HSPCs) reside in bone marrow (BM) niches, which remain challenging to explore due to limited in vivo accessibility and constraints with humanized animal models. Several in vitro systems have been established to culture patient-derived HSPCs in specific microenvironments, but they do not fully recapitulate the complex features of native bone marrow. Our group previously reported that human osteoblastic BM niches (O-N), engineered by culturing mesenchymal stromal cells within three-dimensional (3D) porous scaffolds under perfusion flow in a bioreactor system, are capable of maintaining, expanding, and functionally regulating healthy human cord blood-derived HSPCs. Here, we first demonstrate that this 3D O-N can sustain malignant CD34⁺ cells from acute myeloid leukemia (AML) and myeloproliferative neoplasm patients for up to 3 wk. Human malignant cells distributed in the bioreactor system mimicking the spatial distribution found in native BM tissue, where most HSPCs remain linked to the niches and mature cells are released to the circulation. Using human adipose tissue-derived stromal vascular fraction cells, we then generated a stromal-vascular niche and demonstrated that O-N and stromal-vascular niche differentially regulate leukemic UCSD-AML1 cell expansion, immunophenotype, and response to chemotherapy. The developed system offers a unique platform to investigate human leukemogenesis and response to drugs in customized environments, mimicking defined features of native hematopoietic niches and compatible with the establishment of personalized settings.

Adult bone marrow (BM) stem cell niches are natural blood factories in which BM mesenchymal stromal cells (MSC-BM) and their extracellular matrices interact with hematopoietic stem and progenitor cells (HSPCs) to ensure their maintenance, self-renewal, and differentiation (1, 2). The composition of these BM niches is critical to determine HSPC function. In the murine system, quiescent HSPCs were originally found close to bone surfaces in osteoblastic niches (3–5), but subsequent studies showed that the vast majority of HSPCs locate in niches close to the sinusoidal vasculature (6). These so-called perivascular niches host active HSPCs that circulate between the BM and the bloodstream (7, 8). BM microenvironments also contribute to the development of hematopoietic malignancies (9, 10). Acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN) are clonal hematopoietic malignancies characterized by overproduction of neoplastic hematopoietic cells (11, 12), which evolve and hijack BM microenvironments to create supportive niches for leukemic stem cells (13–19). In contrast to their murine counterparts, human adult BM niches (20) have remained largely unexplored due to the limited accessibility.Multiple humanized animal models have been generated in the last years to model hematological disorders (21), including recently developed patient-derived xenograft models with human BM niche-forming cells (22). Alternatively, human MSC-BM–coated scaffolds have been implanted in mice to generate humanized bone organs capable of hosting healthy murine and human HSPCs (23–26), but also patient-derived leukemic cells (27–30). Nevertheless, none of these in vivo approaches allows studying human malignant HSPCs over prolonged time under controlled settings. In vitro models mimicking the complexity of human BM would represent a solution to investigate the mechanisms of leukemogenesis, while bypassing the ethical concerns associated with the use of experimental animals (31). Some two-dimensional (2D) coculture systems were reported to maintain self-renewing AML progenitor cells for several weeks (from 3 up to 24 wk); these are based on cocultures with either murine (MS-5) or human (Saos-2, HS-5) cell lines (32–34), or human undifferentiated/adipogenic MSC-BM (35, 36). However, they do not recapitulate the complex, specialized signals and cell interactions of native human BM microenvironments. Recently, a three-dimensional (3D) hydrogel triculture system including primary AML cells, MSC-BM, and human umbilical vein endothelial cells (HUVEC) was proposed to model cell–cell interactions in the AML vascular niche (37). Despite introducing the vascular component, this system is based on undifferentiated MSC-BM and thus lacks important features from mature BM niches (e.g., osteoblastic cells and matrix). Although few 3D systems have been developed to model multiple myeloma (38, 39), they are based on classic static cultures and thus, they cannot recapitulate features such as BM interstitial flow and hematopoietic cell circulation. Therefore, 3D multicellular niches mimicking the complexity of human BM niches are not available to culture patient-derived malignant HSPCs for at least 1 wk.We have previously developed a bioreactor-based 3D culture system for MSC-BM that can be used to model in vitro human BM stem cell niches (40). The system enables perfusion in alternate directions of MSC-BM suspensions directly through the pores of 3D hydroxyapatite scaffolds, mimicking the mineral component of trabecular bone. The resulting constructs, recapitulating features of a native human osteoblastic BM niche (O-N), can be further perfused with umbilical cord blood (CB) HSPCs, which establish direct interactions with the O-N and thereby maintain functional properties (41). The culture model offers the unprecedented opportunity to investigate in an in vitro system not only the growth/differentiation of HSPCs within a solid 3D niche, but also the exit of defined subpopulations of HSPCs or their differentiated progenies from the niche. Indeed, hematopoietic cells distributed differentially in the two compartments: while HSPCs remained within the O-N, committed blood cells were released into the liquid phase (supernatant) (41). This system could thus be used to investigate the effects of extrinsic factors (noncell autonomous) on HSPCs, as well as for disease modeling (42).In this study, we aimed at exploiting 3D biomimetic niches engineered in perfusion bioreactors for culturing patient-derived blood cancer cells in fully human microenvironments. First, we assessed the culture of patient-derived AML and MPN CD34⁺ cells in engineered O-N niches for up to 3 wk. We investigated the impact of O-N on cell expansion, immunophenotype, gene expression, and distribution between the bioreactor compartments. Then, we tested the customization potential of our approach by engineering a stromal-vascular niche (SV-N) using human adipose tissue-derived stromal vascular fraction (SVF) cells, leading to stromal tissues enriched in endothelial cells and pericytes (43). We compared O-N and SV-N for culturing leukemic UCSD-AML1 cells and studied the influence of the different microenvironments on leukemic cell expansion, immunophenotype, gene expression, and response to gold-standard chemotherapy (Ara-C; cytarabine), the latter in comparison to classic 2D cultures.     

Macrophage heterogeneity in atherosclerosis: A matter of context

During atherogenesis, plaque macrophages take up and process deposited lipids, trigger inflammation, and form necrotic cores. The traditional inflammatory/anti-inflammatory paradigm has proven insufficient in explaining their complex disease-driving mechanisms. Instead, we now appreciate that macrophages exhibit remarkable heterogeneity and functional specialization in various pathological contexts, including atherosclerosis. Technical advances for studying individual cells, especially single-cell RNA sequencing, indeed allowed to identify novel macrophage subsets in both murine and human atherosclerosis, highlighting the existence of diverse macrophage activation states throughout pathogenesis. In addition, recent studies highlighted the role of the local microenvironment in shaping the macrophages’ phenotype and function. However, this remains largely undescribed in the context of atherosclerosis. In this review we explore the origins of macrophages and their functional specialization, shedding light on the diverse sources of macrophage accumulation in the atherosclerotic plaque. Next, we discuss the phenotypic diversity observed in both murine and human atherosclerosis, elucidating their distinct functions and spatial distribution within plaques. Finally, we highlight the importance of the local microenvironment in both phenotypic and functional specialization of macrophages in atherosclerosis and elaborate on the need for spatial multiomics approaches to provide a better understanding of the different macrophage subsets’ roles in the pathogenesis of atherosclerosis.     

Psychiatry in a Dish: Stem Cells and Brain Organoids Modeling Autism Spectrum Disorders

Autism spectrum disorders are a group of pervasive neurodevelopmental conditions with heterogeneous etiology, characterized by deficits in social cognition, communication, and behavioral flexibility. Despite an increasing scientific effort to find the pathophysiological explanations for the disease, the neurobiological links remain unclear. A large amount of evidence suggests that pathological processes taking place in early embryonic neurodevelopment might be responsible for later manifestation of autistic symptoms. This dysfunctional development includes altered maturation/differentiation processes, disturbances in cell–cell communication, and an unbalanced ratio between certain neuronal populations. All those processes are highly dependent on the interconnectivity and three-dimensional organizations of the brain. Moreover, in order to gain a deeper understanding of the complex neurobiology of autism spectrum disorders, valid disease models are pivotal. Induced pluripotent stem cells could potentially help to elucidate the complex mechanisms of the disease and lead to the development of more effective individualized treatment. The induced pluripotent stem cells approach allows comparison between the development of various cellular phenotypes generated from cell lines of patients and healthy individuals. A newly advanced organoid technology makes it possible to create three-dimensional in vitro models of brain development and structural interconnectivity, based on induced pluripotent stem cells derived from the respective individuals. The biggest challenge for modeling psychiatric diseases in vitro is finding and establishing the link between cellular and molecular findings with the clinical symptoms, and this review aims to give an overview over the feasibility and applicability of this new tissue engineering tool in psychiatry.     

Drosophila ClC-a is required in glia of the stem cell niche for proper neurogenesis and wiring of neural circuits

Glial cells form part of the neural stem cell niche and express a wide variety of ion channels; however, the contribution of these channels to nervous system development is poorly understood. We explored the function of the Drosophila ClC-a chloride channel, since its mammalian ortholog CLCN2 is expressed in glial cells, and defective channel function results in leukodystrophies, which in humans are accompanied by cognitive impairment. We found that ClC-a was expressed in the niche in cortex glia, which are closely associated with neurogenic tissues. Characterization of loss-of-function ClC-a mutants revealed that these animals had smaller brains and widespread wiring defects. We showed that ClC-a is required in cortex glia for neurogenesis in neuroepithelia and neuroblasts, and identified defects in a neuroblast lineage that generates guidepost glial cells essential for photoreceptor axon guidance. We propose that glia-mediated ionic homeostasis could nonautonomously affect neurogenesis, and consequently, the correct assembly of neural circuits.     

The 150 most important questions in cancer research and clinical oncology series:questions 50-56

Since the beginning of 2017,Chinese Journal of Cancer has published a series of important questions in cancer research and clinical oncology,which sparkle diverse thoughts,interesting communications,and potential collaborations among researchers all over the world.In this article,seven more questions are presented as followed.Question 50.When tumor cells spread from primary site to distant sites,are they required to be "trained" or "armed" in the bone marrow niche prior to colonizing soft tissues? Question 51.Are there tipping points during cancer progression which can be identified for manipulation? Question 52.Can we replace molecular biomarkers by network biomarkers? Question 53.Are conventional inhibitors of key cellular processes such as cell proliferation and differentiation more effective than targeted chemotherapeutics that antagonize the downstream cell signaling network via cell-surface receptors such as epidermal growth factor receptor (EGFR),vascular endothelial growth factor receptor (VEGFR) and c-Met,or intraceilular receptors such as androgen receptor (AR) and estrogen receptor (ER),by drugs like erlotinib,sunitinib and cabozantinib,or enzalutamide and tomoxifen? Question 54.How can we robustly identify the candidate causal event of somatic genome alteration (SGA) by using computational approach? Question 55.How can we systematically reveal the immune evasion mechanism exploited by each tumor and utilize such information to guide targeted therapy to restore immune sensitivity? Question 56.Can the nasopharyngeal carcinoma (NPC) patients with sarcomatoid carcinoma (SC) subtype benefit from more specific targeted therapy?     

干细胞微环境的体外模拟

背景：细胞外基质是组织微环境的重要组分,为维持细胞功能提供了关键的生化和物理信号。 目的：对体外模拟骨髓干细胞微环境的研究进展进行综述,着重于介绍体外再造细胞外基质。 方法：应用计算机检索Pubmed (1999-01/2010-10)、Embase (1999-01/2010-10)数据库中关于干细胞微环境的文章,以“mesenchymal stem cells; extracellular matrix; niche; growth factor modulation; culture in vitro”为检索词进行检索。初检得到321篇文献,根据纳入标准选择关于干细胞微环境的23篇文献进行综述。 结果与结论：骨髓源性的细胞外基质培养体系能保持间充质干细胞的特性,对纯化这些细胞和形成更多数量的同质间充质干细胞提供了便利。使用3D合成材料建立一个独特的骨架,包被组织特异性的间充质干细胞蛋白,并添加正确的生长因子组合,有利于调控间充质干细胞的分化方向。     

Immunophenotyping of the human bulge region: the quest to define useful in situ markers for human epithelial hair follicle stem cells and their niche

Abstract:  Since the discovery of epithelial hair follicle stem cells (eHFSCs) in the bulge of human hair follicles (HFs) an important quest has started: to define useful markers. In the current study, we contribute to this by critically evaluating corresponding published immunoreactivity (IR) patterns, and by attempting to identify markers for the in situ identification of human eHFSCs and their niche. For this, human scalp skin cryosections of at least five different individuals were examined, employing standard immunohistology as well as increased sensitivity methods. Defined reference areas were compared by quantitative immunohistochemistry for the relative intensity of their specific IR.
According to our experience, the most useful positive markers for human bulge cells turned out to be cytokeratin 15, cytokeratin 19 and CD200, but were not exclusive, while β1 integrin and Lhx2 IR were not upregulated by human bulge keratinocytes. Absent IR for CD34, connexin43 and nestin on human bulge cells may be exploited as negative markers. α6 integrin, fibronectin, nidogen, fibrillin-1 and latent transforming growth factor (TGF)-beta-binding protein-1 were expressed throughout the connective tissue sheath of human HFs. On the other hand, tenascin-C was upregulated in the bulge and may thus constitute a component of the bulge stem cell niche of human HFs.
These immunophenotyping results shed further light on the in situ expression patterns of claimed follicular 'stem cell markers' and suggest that not a single marker alone but only the use of a limited corresponding panel of positive and negative markers may offer a reasonable and pragmatic compromise for identifying human bulge stem cells in situ .     

Emerging evidence of the molecular landscape specific for hematogenous metastasis from gastric cancer

Gastric cancer (GC) is one of the most frequently diagnosed cancers in the world. Most GC patients are diagnosed when the cancer is in an advanced stage, and consequently, some develop metastatic lesions that generally cause cancer-related death. Metastasis establishment is affected by various conditions, such as tumor location, hemodynamics and organotropism. While digestive cancers may share a primary site, certain cases develop hematogenous metastasis with the absence of peritoneal metastasis, and vice versa. Numerous studies have revealed the clinicopathological risk factors for hematogenous metastasis from GC, such as vascular invasion, advanced age, differentiation, Borrmann type 1 or 2 and expansive growth. Recently, molecular mechanisms that contribute to metastatic site determination have been elucidated by advanced molecular biological techniques. Investigating the molecules that specifically participate in metastasis establishment in distinct secondary organs will lead to the development of novel biomarkers for patient stratification according to their metastatic risk and strategies for preventing and treating distinct metastases. We reviewed articles related to the molecular landscape of hematogenous metastasis from GC.     

干细胞niche与心肌细胞分化的调控机制研究

干细胞是一类具有自我更新和多向分化潜能的特殊细胞。研究发现,干细胞的生物学行为高度依赖于其所处的微环境(niche)。Niche对干细胞的作用分为细胞与细胞之间的直接作用和基质及众多细胞因子对干细胞的间接作用,涉及多条信号转导通路及其相互作用,本文主要介绍骨髓干细胞的niche对间充质干细胞(MSC)的调控,并探讨MSC向心肌细胞分化的调控机制。     

Molecular Checkpoint Decisions Made by Subverted Vascular Niche Transform Indolent Tumor Cells into Chemoresistant Cancer Stem Cells

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