Decellularized spleen matrix for reengineering functional hepatic-like tissue based on bone marrow mesenchymal stem cells

Background and Aims: Decellularized liver matrix (DLM) hold great potential for reconstructing functional hepatic-like tissue (HLT) based on reseeding of hepatocytes or stem cells, but the shortage of liver donors is still an obstacle for potential application. Therefore, an appropriate alternative scaffold is needed to expand the donor pool. In this study, we explored the effectiveness of decellularized spleen matrix (DSM) for culturing of bone marrow mesenchymal stem cells (BMSCs), and promoting differentiation into hepatic-like cells.

Methods: Rats' spleen were harvested for DSM preparation by freezing/thawing and perfusion procedure. Then the mesenchymal stem cells derived from rat bone marrow were reseeded into DSM for dynamic culture and hepatic differentiation by a defined induction protocol.

Results: The research found that DSM preserved a 3-dimensional porous architecture, with native extracellular matrix and vascular network which was similar to DLM. The reseeded BMSCs in DSM differentiated into functional hepatocyte-like cells, evidenced by cytomorphology change, expression of hepatic-associated genes and protein markers, glycogen storage, and indocyanine green uptake. The albumin production (2.74±0.42 vs. 2.07±0.28 pg/cell/day) and urea concentration (75.92±15.64 vs. 52.07±11.46 pg/cell/day) in DSM group were remarkably higher than tissue culture flasks (TCF) group over the same differentiation period, P< 0.05.

Conclusion: This present study demonstrated that DSM might have considerable potential in fabricating hepatic-like tissue, particularly because it can facilitate hepatic differentiation of BMSCs which exhibited higher level and more stable functions.     

Identification of a clonally expanding haematopoietic compartment in bone marrow

In mammals, postnatal haematopoiesis occurs in the bone marrow (BM) and involves specialized microenvironments controlling haematopoietic stem cell (HSC) behaviour and, in particular, stem cell dormancy and self‐renewal. While these processes have been linked to a number of different stromal cell types and signalling pathways, it is currently unclear whether BM has a homogenous architecture devoid of structural and functional partitions. Here, we show with genetic labelling techniques, high‐resolution imaging and functional experiments in mice that the periphery of the adult BM cavity harbours previously unrecognized compartments with distinct properties. These units, which we have termed hemospheres, were composed of endothelial, haematopoietic and mesenchymal cells, were enriched in CD150+ CD48? putative HSCs, and enabled rapid haematopoietic cell proliferation and clonal expansion. Inducible gene targeting of the receptor tyrosine kinase VEGFR2 in endothelial cells disrupted hemospheres and, concomitantly, reduced the number of CD150+ CD48? cells. Our results identify a previously unrecognized, vessel‐associated BM compartment with a specific localization and properties distinct from the marrow cavity.     

Frequent harvesting from perfused bone marrow cultures results in increased overall cell and progenitor expansion

The establishment of prolific long-term human bone marrow cultures has led to the development of hematopoietic bioreactor systems. A single batch expansion of bone marrow mononuclear cell populations leads to a 10- to 30-fold increase in total cell number and in the number of colony forming units-granulocyte/macrophage (CFU-GMs), and a four- to tenfold increase in the number of long-term culture initiating cells (LTC-ICs). In principle, unlimited expansion of cells should be attainable from a pool of stem cells if all the necessary requirements leading to stem cell maintenance and division are met. In this article, we take the first step toward the identification of factors that limit single batch expansion of ex vivo bone marrow cells in perfusion-based bioreactor systems. One possible constraint is the size of the growth surface area required. This constraint can be overcome by harvesting half the cell population periodically. We found that harvesting cells every 3 to 4 days, beginning on day 11 of culture, led to an extended growth period. Overall calculated cell expansion exceeded 100-fold and the CFU-GM expansion exceeded 30-fold over a 27-day period. These calculated values are based on growth that could be obtained from the harvested cell population. Growth of the adherent cell layer was stable, whereas the nonadherent cell population diminished with increasing number of passages. These results show that the bioreactor protocols published to date are suboptimal for long-term cultivation, and that further definition and refinement is likely to lead to even greater expansion of hematopoietic cell populations obtained from bone marrow. More importantly, these results show that the LTC-IC measured during the single pass expansion do have further expansion potential that can be realized by frequent harvesting. Finally, the present culture conditions provide a basis for an assay system for the identifications provide a basis for an assay system for the identification of the factors that determine the long-term maintenance and replication of human stem cells ex vivo. (c) 1994 John Wiley & Sons, Inc.     

Recapitulative haematopoietic development of human pluripotent stem cells in the absence of exogenous haematopoietic cytokines

To improve the recapitulative quality of human pluripotent stem cell (hPSC) differentiation, we removed exogenous haematopoietic cytokines from the defined differentiation system. Here, we show that endogenous stimuli and VEGF are sufficient to induce robust hPSC-derived haematopoiesis, intensive generation of haematopoietic progenitors, maturation of blood cells and the emergence of definitive precursor cells including those that phenotypically identical to early human embryonic haematopoietic stem cells (HSCs). Moreover, the cytokine-free system produces significantly higher numbers of haematopoietic progenitors compared to the published protocols. The removal of cytokines revealed a broad developmental potential of the early blood cells, stabilized the hPSC-derived definitive precursors and led to spontaneous activation of inflammatory signalling. Our cytokine-free protocol is simple, efficient, reproducible and applicable for embryonic stem cells (ESCs) and induced PSCs. The spectrum of recapitulative features of the novel protocol makes the cytokine-free differentiation a preferred model for studying the early human haematopoietic development.     

Cyclin A1 regulates the interactions between mouse haematopoietic stem and progenitor cells and their niches

It remains poorly understood how the haematopoietic stem/progenitor cells (HSPC) are attracted to their niches and the functional consequences of such interaction. In the present study, we show that the cell cycle regulator cyclin A1 in association with vascular endothelial growth factor receptor 1 (VEGFR1), is required for HSPC and their niches to maintain their function and proper interaction. In the absence of cyclin A1, the HSPC in the BM are increased in their frequency and display an increased migratory and homing ability. Concomitantly, the ability of the endosteal and central BM niche zones to attract and home the wild-type HSPC is significantly reduced in cyclin A1-null mice as compared to the wild-type controls. The impaired proliferation and homing of HSPC in the BM of cyclin A1-null mice are attributed to the increased density of microvessels in the endosteal and central BM niche zones, which is associated with the increased VEGFR1 expression. Thus, modulation of cyclin A1 and VEGFR1 in HSPC and their niches may provide new insights into therapeutic approaches.     

Icariin promotes osteogenic differentiation of bone marrow stromal cells and prevents bone loss in OVX mice via activating autophagy

Osteoporosis (OP) results from the impaired function of endogenous bone marrow mesenchymal stem cells (BMSCs). Icariin (ICA) has shown potential osteoprotective effects. However, the molecular mechanism for the anabolic action of ICA remains largely unknown. The objective of the present study is to investigate whether ICA prevents bone loss by acting on BMSCs via affecting the level of autophagy after ovariectomy (OVX). The BMSCs were extracted from BALB/c mice treated with ICA, chloroquine (CQ, an autophagy inhibitor) or ICA + CQ. The OVX mice were injected with ICA, CQ, or ICA + CQ for 1 month. We performed Alizarin Red staining and alkaline phosphatase staining to detect osteogenic differentiation of BMSCs. Micro-CT, hematoxylin and eosin staining, Oil Red O staining, and tartrate-resistant acid phosphatase staining were used to assess the bone mass, lipid droplets and osteoclasts in femurs. Autophagy activity in BMSCs from different groups was evaluated by Western blot analysis. The osteogenic differentiation of BMSCs from OVX-induced OP mice was decreased. Treatment with ICA reduced bone loss and formation of osteoclasts and increased osteogenic differentiation of BMSCs in vitro and vivo. In addition, autophagy was enhanced in BMSCs of OVX mice treated with ICA. Our results indicate that ICA prevents OVX-induced bone loss possibly by strengthening the osteogenic differentiation of BMSCs via increasing autophagic activity.     

Differentiation of embryonic stem cells in adult bone marrow

Embryonic stem cells (ESCs) are a potential source of generating transplantable hematopoietic stem and progenitor cells, which in turn can serve as "seed" cells for hematopoietic regeneration. In this study, we aimed to gauge the ability of mouse ESCs directly differentiating into hematopoietic cells in adult bone marrow (BM). To this end, we first derived a new mouse ESC line that constitutively expressed the green fluorescent protein (GFP) and then injected the ESCs into syngeneic BM via intra-tibia. The progeny of the transplanted ESCs were then analyzed at different time points after transplantation. Notably, however, most injected ESCs differentiated into non-hematopoietic cells in the BM whereas only a minority of the cells acquired hematopoietic cell surface markers. This study provides a strategy for evaluating the differentiation potential of ESCs in the BM micro-environment, thereby having important implications for the physiological maintenance and potential therapeutic applications of ESCs.     

孕期应激对子代大鼠骨髓淋巴干细胞发育的影响

孕期应激对子代产生的影响是多方面的,这种影响是复杂的。研究表明,出生前的应激经历可导致出生后子代长期的免疫功能改变。这些改变追其根源与骨髓淋巴干细胞的改变有关。本文综述了大鼠孕期经历应激的子代骨髓淋巴干细胞所受的影响及免疫系统的相关改变,并根据现有的研究提出假说,为进一步研究孕期应激导致子代免疫系统改变的机理研究提供新的思路。     

Characterization of effector cells with anti-Candida activity obtained from murine bone marrow cells cultured in the presence of rhG-CSF: comparison between normal and CY-treated mice

Bone marrow cells (BMC) obtained from normal and cyclophosphamide (CY)-treated mice were cultured in the presence of recombinant human granulocyte-colony stimulating factor (rhG-CSF) and their effector cell activities inhibiting growth of C. albicans were examined. When BMC from CY-treated mice were preincubated with 0.05 ng/ml of rhG-CSF, effector cells with enhanced anti-C. albicans activity were recovered in the adherent cell population, whereas anti-C. albicans activity of BMC from normal mice was found in the non-adherent cell population. During culture without the presence of rhG-CSF, nonadherent BMC, seemingly granulocytes, from normal mice showed apoptotic change, but addition of rhGCSF clearly inhibited this change. On the other hand, when BMC from CY-treated mice were cultured with rhG-CSF, adherent cells as the main effector had the appearance of monocytes. These differences between the effectors with anti-C. albicans activity obtained from normal and CY-treated mice are discussed.     

Bone and bone marrow disruption by endocrine-active substances

Bone is a multifaceted dynamic tissue, involved in mobility, mineral metabolism, and mesenchymal or stromal and hematopoietic progenitor or stem cells breading. Recently, an endocrine role has been attributed to bone due to its ability to produce at least two hormones (osteocalcin and fibroblast growth factor 23) and to participate directly or indirectly in leptin, insulin, estrogens, and serotonin signaling; regulation; and action. Also, bearing in mind the enormous amounts of substances secreted by the different bone marrow cell types, it becomes understandable the contribution of bone tissue to systemic homeostasis. Besides, bone is a well-known estrogen-responsive tissue, reacting to environmental influences. Thus, it has been coined as a critical target of environmental xenoestrogens, known as endocrine-disrupting chemicals (EDCs). The exposure to EDCs results to disruption or imbalance of the systemic hormonal regulation of the skeleton including bone modeling and remodeling, local hormones, and cytokine or chemokine release. The present report highlights the harmful EDCs effects on bone tissue and provides up-to-date information of xenoestrogen action on proliferation, maturation, and homing of bone marrow inhabitants.     

孕期应激对子代大鼠骨髓淋巴干细胞发育的影响

孕期应激对子代产生的影响是多方面的,这种影响是复杂的。研究表明,出生前的应激经历可导致出生后子代长期的免疫功能改变。这些改变追其根源与骨髓淋巴干细胞的改变有关。本文综述了大鼠孕期经历应激的子代骨髓淋巴干细胞所受的影响及免疫系统的相关改变,并根据现有的研究提出假说,为进一步研究孕期应激导致子代免疫系统改变的机理研究提供新的思路。     

Cathepsin K deficiency promotes alveolar bone regeneration by promoting jaw bone marrow mesenchymal stem cells proliferation and differentiation via glycolysis pathway

ObjectivesTo clarify the possible role and mechanism of Cathepsin K (CTSK) in alveolar bone regeneration mediated by jaw bone marrow mesenchymal stem cells (JBMMSC).Materials and MethodsTooth extraction models of Ctsk knockout mice (Ctsk ^‐/‐) and their wildtype (WT) littermates were used to investigate the effect of CTSK on alveolar bone regeneration. The influences of deletion or inhibition of CTSK by odanacatib (ODN) on proliferation and osteogenic differentiation of JBMMSC were assessed by CCK‐8, Western blot and alizarin red staining. To explore the differently expressed genes, RNA from WT and Ctsk^‐/‐ JBMMSC was sent to RNA‐seq. ECAR, glucose consumption and lactate production were measured to identify the effect of Ctsk deficiency or inhibition on glycolysis. At last, we explored whether Ctsk deficiency or inhibition promoted JBMMSC proliferation and osteogenic differentiation through glycolysis.ResultsWe found out that Ctsk knockout could promote alveolar bone regeneration in vivo. In vitro, we confirmed that both Ctsk knockout and inhibition by ODN could promote proliferation of JBMMSC, up‐regulate expression of Runx2 and ALP, and enhance matrix mineralization. RNA‐seq results showed that coding genes of key enzymes in glycolysis were significantly up‐regulated in Ctsk^‐/‐ JBMMSC, and Ctsk deficiency or inhibition could promote glycolysis in JBMMSC. After blocking glycolysis by 3PO, the effect of Ctsk deficiency or inhibition on JBMMSC’s regeneration was blocked subsequently.ConclusionsOur findings revealed that Ctsk knockout or inhibition could promote alveolar bone regeneration by enhancing JBMMSC regeneration via glycolysis. These results shed new lights on the regulatory mechanism of CTSK on bone regeneration.     

小鼠骨髓内皮细胞分泌的细胞因子对造血干/祖细胞增殖的影响

应用小鼠骨髓内皮细胞株细胞传代培养,收集无血清条件培养液（ｍＢＭＥＣ－ＣＭ）,经超滤分成大于１０ｋＤ和小于１０ｋＤ两组分,分别观察两组分的ｍＢＭＥＣ－ＣＭ对小鼠骨髓造血干／祖细胞ＣＦＵ－ＧＭ,ＨＰＰ－ＣＦＣ,ＣＦＵ－Ｅ,ＢＦＵ－Ｅ和ＣＦＵ－Ｍｅｇ的影响。结果表明：含分子量大于１０ｋＤ物质的ｍＢＭＥＣ－ＣＭ的保留液能明显刺激ＣＦＵ－ＧＭ,ＨＰＰ－ＣＦＣ,ＣＦＵ－Ｅ,ＢＦＵ－Ｅ和ＣＦＵ－Ｍｅｇ生长;     

Local signals in stem cell-based bone marrow regeneration

The cellular basis of bone marrow （BM） tissue development and regeneration is mediated through hematopoietic stem cells （HSCs） and mesenchymal stem cells （MSCs）. Local interplays between hematopoietic cells and BM stromal cells （BMSCs） determine the reconstitution of hematopoiesis after myelosuppression. Here we review the BM local signals in control of BM regeneration after insults. Hematopoietic growth factors （HGFs） and cytokines produced by BMSCs are primary factors in regulation ofBM hematopoiesis. Morphogens which are critical to early embryo development in multiple species have been added to the family of HSCs regulators, including families of Wnt proteins, Notch ligands, BMPs, and Hedgehogs. Global gene expression analysis of HSCs and BMSCs has begun to reveal signature groups of genes for both cell types. More importantly, analysis of global gene expression coupled with biochemical and biological studies of local signals during BM regeneration have strongly suggested that HGFs and cytokines may not be the primary local regulators for BM recovery, rather chemokines （SDF- 1, FGF-4） and angiogenic growth factors （VEGF-A, Ang- 1） play instructive roles in BM reconstitution after myelosuppression. A new direction of management of BM toxicity is emerging from the identification of BM regenerative regulators.     

The role of novel and known extracellular matrix and adhesion molecules in the homeostatic and regenerative bone marrow microenvironment

Maintenance of haematopoietic stem cells and differentiation of committed progenitors occurs in highly specialized niches. The interactions of haematopoietic stem and progenitor cells (HSPCs) with cells, growth factors and extracellular matrix (ECM) components of the bone marrow (BM) microenvironment control homeostasis of HSPCs. We only start to understand the complexity of the haematopoietic niche(s) that comprises endosteal, arterial, sinusoidal, mesenchymal and neuronal components. These distinct niches produce a broad range of soluble factors and adhesion molecules that modulate HSPC fate during normal hematopoiesis and BM regeneration. Adhesive interactions between HSPCs and the microenvironment will influence their localization and differentiation potential. In this review we highlight the current understanding of the functional role of ECM- and adhesion (regulating) molecules in the haematopoietic niche during homeostatic and regenerative hematopoiesis. This knowledge may lead to the improvement of current cellular therapies and more efficient development of future cellular products.     

The role of novel and known extracellular matrix and adhesion molecules in the homeostatic and regenerative bone marrow microenvironment

Maintenance of haematopoietic stem cells and differentiation of committed progenitors occurs in highly specialized niches. The interactions of haematopoietic stem and progenitor cells (HSPCs) with cells, growth factors and extracellular matrix (ECM) components of the bone marrow (BM) microenvironment control homeostasis of HSPCs. We only start to understand the complexity of the haematopoietic niche(s) that comprises endosteal, arterial, sinusoidal, mesenchymal and neuronal components. These distinct niches produce a broad range of soluble factors and adhesion molecules that modulate HSPC fate during normal hematopoiesis and BM regeneration. Adhesive interactions between HSPCs and the microenvironment will influence their localization and differentiation potential. In this review we highlight the current understanding of the functional role of ECM- and adhesion (regulating) molecules in the haematopoietic niche during homeostatic and regenerative hematopoiesis. This knowledge may lead to the improvement of current cellular therapies and more efficient development of future cellular products.     

Transplantation with bone marrow stromal cells promotes wound healing under chemotherapy through altering phenotypes

Stem cell transplantation is a promising strategy for delayed wound healing caused by chemotherapy. However, the fate of stem cells under chemotherapy has not been fully elucidated. Herein we characterized human fetal bone marrow stromal cells (hBMSCs) during wound healing in mice treated with cyclophosphamide (CTX). The isolated hBMSCs expressed the phenotype of CD11b(low)/CD14(low)/CD34(low)/CD45(low)/CD29(high)/CD44(high)/CD90(high)/CD105(high)/CD146(high)/STRO-1(low). Following in vitro exposure to CTX, hBMSCs showed decreased cell growth in a dose- and time-dependent manner, accompanied by increased expressions of collagen-I/III, and CD31. After transplantation, wounds closed as early as 8 days and were positive for α-smooth muscle actin (α-SMA), implicating the enhanced re-epithelialization and wound contraction. Moreover, proliferating cell nuclear antigen (PCNA) and CD31 showed co-localization with α-SMA, suggesting the differentiation of hBMSCs into epithelial cells and myofibroblasts/fibroblasts. Taken together, our results indicate hBMSCs can accelerate wound healing under chemotherapy through altering their phenotypes.     

Proteomic profiling of distinct clonal populations of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi‐lineage differentiation. The molecular mechanisms governing MSC self‐renewal and differentiation remain largely unknown. The development of sophisticated techniques, in particular clinical proteomics, has enabled researchers in various fields to identify and characterize cell specific biomarkers for therapeutic purposes. This study seeks to understand the cellular and sub‐cellular processes responsible for the existence of stem cell populations in bone marrow samples by revealing the whole cell proteome of the clonal cultures of bone marrow‐derived MSCs (BMSCs). Protein profiling of the MSC clonal populations was conducted by Two‐Dimensional Liquid Chromatography/Matrix‐Assisted Laser Desorption/Ionisation (MALDI) Mass Spectrometry (MS). A total of 83 proteins were identified with high confidence of which 11 showed differential expression between subpopulations, which included cytoskeletal and structural proteins, calcium binding proteins, cytokinetic proteins, and members of the intermediate filament family. This study generated a proteome reference map of BMSCs from the clonal populations, which will be valuable to better understand the underlying mechanism of BMSC self‐renewal and differentiation. J. Cell. Biochem. 106: 776–786, 2009. © 2009 Wiley‐Liss, Inc.     

骨髓基质干细胞的分离纯化及培养

目的 建立骨髓基质干细胞(MSCs)良好的分离纯化和培养方法。方法 将小鼠骨髓基质干细胞自殷骨中分离,应用贴壁选择法结合细胞克隆挑选法进行分离纯化,应用细胞生长因子(EGF和PDGF-BB)刺激法进行MSCs的体外培养和传代,倒置显微镜下观察分离培养的细胞并照像记录。结果,培养获得了纯化的呈梭形成纤雏样细胞的骨髓基质干细胞。在生长因子EGF和PDGF-BB的共同作用下,传代MSCs生长旺盛,形态均一。结论 该方法是简便高效的骨髓基质干细胞的分离纯化和培养方法。