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Regulation of hemopoietic stem cell proliferation in long term bone marrow cultures
Abstract
The development of a suitable bone marrow derived adherent cell population appears to be essential for the prolonged maintenance of haemopoietic stem cells in vitro. When established adherent layers are inoculated with freshly isolated bone marrow cells, proliferation of stem cells (CFU-S) regularly occurs both in the adherent layer and amongst the non-adherent cells. Furthermore, CFU-S present within the adherent layer are able to regenerate both themselves and the "non-adherent" CFU-S. One day after re-feeding the cultures (by removal of half the growth medium and addition of fresh medium) both the "non-adherent" and the "adherent" CFU-S are in a high cycling state (greater than 40% kill with 3HTdR). This proportion decreases with time of re-feeding and 5-7 days later the majority of "adherent" and "non-adherent" CFU-S are in a low cycling state ( less than 10% 3HTdR kill). Following a further re-feeding, CFU-S again enter a high cycling state.
... Later studies by Dexter and colleagues in the mid 1970s resulted in the establishment of a culture system that maintained primitive mouse hematopoietic progenitor cells in vitro [60,61]. From these observations the authors conclude that the maintenance of hematopoiesis in vitro was dependent on an adherent layer of marrow derived cells whose composition reflected the phenotypic diversity of the stromal elements in the BM in vivo [61]. ...
... Later studies by Dexter and colleagues in the mid 1970s resulted in the establishment of a culture system that maintained primitive mouse hematopoietic progenitor cells in vitro [60,61]. From these observations the authors conclude that the maintenance of hematopoiesis in vitro was dependent on an adherent layer of marrow derived cells whose composition reflected the phenotypic diversity of the stromal elements in the BM in vivo [61]. The historical importance of these findings allowed Ray Schofield to formulate his niche hypothesis in which he states that, "the stem cell is seen in association with other cells which determine its behavior. ...
... Such molecules include SCF, Angiopoietin 1, CXCL12 (SCF-1), osteopontin, and Ca 2+ ion. However, while the application of mouse genetics as applied to gain-and loss-of-function studies has resulted in significant discoveries of these genetically determined factors, there still remains a paucity of information and limitations as applied to this approach in identifying the specific cells types responsible for generating these regulatory A second approach taken by many investigators has been to apply the longterm bone marrow culture system first reported by Dexter and colleagues [60,61] as a means to interrogate the heterogeneous microenvironment in vitro. As reviewed in the previous chapters, the bone marrow microenvironment is comprised of a diverse population of hematopoietic macrophage and non-hematopoietic stromal cells. ...
Bone marrow (BM) stromal cells are ascribed two key functions, 1) stem cells for non-hematopoietic tissues (MSC) and 2) as components of the hematopoietic stem cell niche. Current approaches studying the stromal cell system in the mouse are complicated by the low yield of clonogenic progenitors (CFU-F). Given the perivascular location of MSC in BM, we developed an alternative methodology to isolate MSC from mBM. An intact ‘plug’ of bone marrow is expelled from bones and enzymatically disaggregated to yield a single cell suspension. The recovery of CFU-F (1917.95+199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32+1.9) by at least 2 orders of magnitude (P
... To evaluate granulocytic differentiation, long-term bone marrow culture (LTMBC) was employed following Dexter's (1977) 27 protocol as a way to sustain HSC and myeloid populations in vitro. The percentage of the LSK population was not affected by rAnxA1 treatment (Fig. 6a). ...
... Long-term bone marrow cultures (LTBMCs) were prepared as previously described by Dexter et al. 27 with some modifications. Briefly, 10 6 total bone marrow cells were plated into 12-well plates and fed weekly with Iscove's modified Dulbecco's medium (IMDM) supplemented with 12.5% foetal bovine serum, 12.5% horse serum (StemCell Technologies, Canada) and 1 μM hydrocortisone (Sigma Aldrich, USA). ...
Annexin A1 (AnxA1) modulates neutrophil life span and bone marrow/blood cell trafficking thorough activation of formyl-peptide receptors (FPRs). Here, we investigated the effect of exogenous AnxA1 on haematopoiesis in the mouse. Treatment of C57BL/6 mice with recombinant AnxA1 (rAnxA1) reduced the granulocyte–macrophage progenitor (GMP) population in the bone marrow, enhanced the number of mature granulocytes Gr-1+Mac-1+ in the bone marrow as well as peripheral granulocytic neutrophils and increased expression of mitotic cyclin B1 on hematopoietic stem cells (HSCs)/progenitor cells (Lin−Sca-1+c-Kit+: LSK). These effects were abolished by simultaneous treatment with Boc-2, an FPR pan-antagonist. In in vitro studies, rAnxA1 reduced both HSC (LSKCD90lowFLK-2−) and GMP populations while enhancing mature cells (Gr1+Mac1+). Moreover, rAnxA1 induced LSK cell proliferation (Ki67+), increasing the percentage of cells in the S/G2/M cell cycle phases and reducing Notch-1 expression. Simultaneous treatment with WRW4, a selective FPR2 antagonist, reversed the in vitro effects elicited by rAnxA1. Treatment of LSK cells with rAnxA1 led to phosphorylation of PCLγ2, PKC, RAS, MEK, and ERK1/2 with increased expression of NFAT2. In long-term bone marrow cultures, rAnxA1 did not alter the percentage of LSK cells but enhanced the Gr-1+Mac-1+ population; treatment with a PLC (U73122), but not with a PKC (GF109203), inhibitor reduced rAnxA1-induced phosphorylation of ERK1/2 and Elk1. Therefore, we identify here rAnxA1 as an inducer of HSC/progenitor cell differentiation, favouring differentiation of the myeloid/granulocytic lineage, via Ca2+/MAPK signalling transduction pathways.
... Within the patient, AML cells reside in a complex microenvironment that includes bone marrow stroma cells and endothelial cells among many cell types. Since the long-term culture for normal hematopoietic cells, first described by Dexter et al. [9], there have been numerous studies employing stroma cell based microenvironment as a surrogate for the bone marrow GML cell niche to study the molecular cross-talk, stem cell maintenance, and therapeutic resistance [10−14]. In this study, we established and characterized a new cell line, OCI-AML-20, which has inv(3) and loss of chromosome 7. ...
... OCI-AML-20 cell line subpopulations On stroma cells, OCI-AML-20 exhibits a semi-adherent growth pattern without the classical cobblestone morphology [9]. To better visualize the interaction between OCI-AML-20 and stroma, we generated GFP-labeled OCI-AML-20 cells and mCherry-labeled OP9 cells. ...
Acute myeloid leukemia (AML) is a complex, heterogeneous disease with variable outcomes following curative intent chemotherapy. AML with inv(3) is a genetic subgroup characterized by a very low response rate to current induction type chemotherapy and thus has among the worst long-term survivorship of the AMLs. Here, we describe OCI-AML-20, a new AML cell line with inv(3) and deletion of chromosome 7; the latter is a common co-occurrence in inv(3) AML. In OCI-AML-20, CD34 expression is maintained and required for repopulation in vitro and in vivo. CD34 expression in OCI-AML-20 shows dependence on the co-culture with stromal cells. Transcriptome analysis indicates that the OCI-AML-20 clusters with other AML patient data sets that have poor prognosis, as well as other AML cell lines, including another inv(3) line, MUTZ-3. OCI-AML-20 is a new cell line resource for studying the biology of inv(3) AML that can be used to identify potential therapies for this poor outcome disease.
... In retrospect, a partial explanation for the benefits of MSC administration traces back to some of the very first observations made with bone marrow stromal cells. In the 1970s, Dexter and colleagues were the first to demonstrate that adherent stromal cells from bone marrow (later identified as MSCs) could sustain the growth, viability, and multipotent status of hematopoietic stem cells in long-term co-cultures that lacked growth factor supplementation [26][27][28][29]. Of particular interest was that the cultures achieved homeostasis with the self-renewal of progenitor cells balanced against the development of committed hematopoietic cells. ...
... The pro-survival effect(s) of the MSC secretome on other cell types was first recognized through studies of long-term bone marrow cultures [26][27][28][29]75] and embryonic cells [76]. Collectively, these cell culture studies provide for an attractive, paracrine-based explanation for the ability of MSCs to promote healing across a broad range of developmentally unrelated tissues and for myriad diseases and injury types. ...
The past decade has seen an explosion of research directed toward better understanding of the mechanisms of mesenchymal stem/stromal cell (MSC) function during rescue and repair of injured organs and tissues. In addition to delineating cell–cell signaling and molecular controls for MSC differentiation, the field has made particular progress in defining several other mechanisms through which administered MSCs can promote tissue rescue/repair. These include: 1) paracrine activity that involves secretion of proteins/peptides and hormones; 2) transfer of mitochondria by way of tunneling nanotubes or microvesicles; and 3) transfer of exosomes or microvesicles containing RNA and other molecules. Improved understanding of MSC function holds great promise for the application of cell therapy and also for the development of powerful cell-derived therapeutics for regenerative medicine. Focusing on these three mechanisms, we discuss MSC-mediated effects on immune cell responses, cell survival, and fibrosis and review recent progress with MSC-based or MSC-derived therapeutics.
... Since the first in vitro description of mixed bone marrow stromal cultures with hematopoietic output in 2D conditions (Dexter, Wright, Krizsa, & Lajtha, 1977), more refined methods have demonstrated the capacity for limited hematopoietic expansion in vitro in highly controlled conditions, often stroma-free, and with potential use for HSPC expansion in clinical transplantation (Bujko, Kucia, Ratajczak, & Ratajczak, 2019;Derakhshani et al., 2019). These methods, however, do not allow the study of HSPC interactions with the niche that regulates their behavior in homeostatic conditions. ...
Although hematopoietic stem cell (HSC) transplantation can restore functional hematopoiesis upon immune or chemotherapy‐induced bone marrow failure, complications often arise during recovery, leading to up to 25% transplant‐related mortality in treated patients. In hematopoietic homeostasis and regeneration, HSCs in the bone marrow give rise to the entirety of cellular blood components. One of the challenges in studying hematopoiesis is the ability to successfully mimic the relationship between the stroma and hematopoietic stem and progenitor cells (HSPCs). This study and the described protocols propose an advantageous method for culturing and assessing stromal hematopoietic support in three dimensions, representing a simplified in vitro model of the bone marrow niche that can be transplanted in vivo by injection. By co‐culturing OP9 bone marrow–derived stromal cells (BMSCs) and cKit ⁺ Sca‐1 ⁺ Lin – (KLS ⁺ ) HSPCs on collagen‐coated carboxymethylcellulose scaffolds for 2 weeks in the absence of cytokines, we established a methodology for in vivo subcutaneous transplantation. With this model we were able to detect early signs of extramedullary hematopoiesis. This work can be useful for studying various stromal cell populations in co‐culture, as well as simple transfer by injection of these scaffolds in vivo for heterotopic regeneration of the marrow microenvironment. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.
[Correction added on May 17, 2022, after first online publication: CSAL funding statement has been added.]
Basic Protocol 1 : Isolation of HSPCs from mice
Basic Protocol 2 : Co‐seeding of HSPCs and BMSCs on collagen‐coated CCMs
Basic Protocol 3 : Maintenance, real‐time imaging, and analysis of co‐seeded scaffolds
Basic Protocol 4 : End‐point analysis of co‐seeded scaffolds using flow cytometry and CFU assays
Basic Protocol 5 : Transplantation of scaffolds by subcutaneous injection
Support Protocol : Preparation of custom scaffold drying device
... Layered co-cultures to model normal haematopoiesis and to culture AML blasts are widely used since Dexter et al. 9 first described it in 1977. It is also well established that BMSCs can provide survival signals to AML cells and protect them from cytotoxic drugs. ...
Between 10-40% of acute myeloid leukaemia (AML) patients are resistant to induction chemotherapy. While patient outcome could be improved by selecting the right therapy, reliable prediction of treatment failure based on cytogenetics, molecular profile or gene expression signatures is still a challenge. The impact of the bone marrow microenvironment (BMM) is increasingly recognised as a reason for this. We have developed and characterised a 3D co-culture of primary AML blasts that is able to model bone marrow-mediated drug resistance. With kinome and secretome studies, we show the complex signalling networks triggered by BMSCs driving AML survival and drug resistance. We show that the developed BMSC-AML co-culture can predict the clinical response to AraC+Dnr therapy with very high accuracy (AUC=0.94). The advantage of this model over more complex pre-clinical AML models is its suitability to be developed into a laboratory diagnostic tool to advance the clinical decision on treatment choice.
... Tissue engineering, as well as plastic surgery, considers MSCs as a promising tool (Yorukoglu et al., 2017;Eun, 2014). Dexter et al. (1977) were one of the first teams to show the ability of MSCs to support growth and survival of hematopoietic stem cells. Later other investigators demonstrated the abilities of MSCs to promote neurotrophic influence and support functional recovery of patients with stroke; MSCs promoted repair of injured cardiac tissue, restoration of knee cartilage, and recovery from other injuries (Aggarwal and Pittenger, 2005;Di Nicola et al., 2002;Shabbir et al., 2010;Centeno et al., 2008;Andrews et al., 2008). ...
... The cargo-bearing sEVs released by hMSCs, for example, contain MSC-associated critical surface markers and signaling molecules characteristic of the parental cell, thereby potentially mediating the therapeutic effects of the MSCs [10]. This notion of MSC paracrinemediated therapeutic effect has been reported in many studies where low cell engraftment after systemic administration of MSCs was described, while clinical effectiveness was still achieved [11][12][13]. ...
Background:
Clinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance.
Methods:
Small EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers.
Results:
Nanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 109/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors.
Conclusion:
The identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.
... Dans les années 1970, un modèle de microenvironnement inductif de l'hématopoïèse a été décrit (revue dans [Trentin, 1989]). En 1977, Dexter a proposé l'utilisation d'une couche de cellules stromales adhérentes pour le maintien des HSC de souris en culture à long terme [Dexter, 1977]. Ces systèmes ont contribué à approfondir le concept de niche par Robert Schofield en 1978. ...
Il est maintenant établi que les cellules souches mésenchymateuses (MSC), résident dans la même niche que les cellules souches hématopoïétiques (HSC), au sein de la moelle osseuse (MO). Il est connu que la pression en O2 (pO2) de la niche est inférieure à la normale, soit moins de 5% pO2 contre 12-15 % pO2 dans le sang artériel. Cette hypoxie a des conséquences sur le métabolisme, en protégeant les cellules contre le stress oxydatif et en favorisant leur caractère multipotent. Notre hypothèse est que les MSC cultivées en hypoxie devraient être plus proches de leur condition physiologique, donc plus multipotentes. Des MSC de la MO humaine ont été cultivées en pO2 de 21% et 5%. Leur morphologie, capacité de différenciation en ostéocytes et adipocytes, et transcriptome ont été comparés à différents passages. Nous avons observé un ralentissement de la prolifération à des temps précoces à pO2 5%, caractérisée par une inhibition de l'expression de gènes impliqués dans la réplication et le cycle cellulaire, puis une augmentation à des passages tardifs. Les gènes codant pour des molécules d'adhérence et de la matrice extracellulaire sont stimulés par l'hypoxie. A des temps tardifs, la capacité de différenciation des MSC est stimulée en hypoxie, les cellules présentent un aspect plus immature et une diminution de synthèse des mitochondries. Surtout, l'hypoxie stimule la synthèse de « gènes de la plasticité » suivant le logiciel « Gene Ontology », et de gènes impliqués dans le développement épithélial et neuronal. En conclusion, la culture des MSC de MO en hypoxie semble plus physiologique et pourrait être utile pour des applications en médecine régénératrice
... .5 La niche hématopoïétique1.5.1 La notion de nicheUn premier modèle de microenvironnement inductif de l'hématopoïèse a été décrit dans les années 1970(Trentin, 1989). Puis, en 1977, Dexter a proposé l'utilisation d'une couche de cellules stromales adhérentes pour le maintien des CSH de souris en culture à long terme(Tm et al., 1977). Toutefois, c'est en 1978, que Robert Schofield a évoqué le concept de niche pour expliquer le microenvironnement limité qui contenait les CSH(Schofield, 1978). ...
Située au CHU de Brabois, la Banque de Sang Placentaire (BSP) de Nancy a ouvert sesportes le 19 novembre 2012. Cette BSP est associée à la Polyclinique Majorelle qui s’occupe durecrutement des mères et du prélèvement des Unités de Sang Placentaire (USP).Le Réseau Français de Sang Placentaire (RFSP), créé en janvier 1999 par l’Agence de laBiomédecine (ABM), a mis en place des critères stricts de sélection des USP mis en banque. Ainsidès leur réception à la BSP, les USP sont mises en production et éliminées pour qu’il ne reste que lesUSP conforme à toutes les spécifications du RFSP. Les causes d’élimination sont très diverses :volume sanguin trop faible, nombre de cellules nucléées totales (CNT) trop faible, nombre de cellulesCD34+ trop faible, contamination microbienne, problème au moment de la congélation… Nous allonsdétailler ici toutes ces causes et voir s’il existe des moyens de les limiter afin d’obtenir un meilleurrendement d’USP inscrites sur le registre national France Greffe de Moelle (FGM) par rapport à tousles prélèvements effectués.Deux facteurs prioritaires sont pris en compte lors de la cession d’une USP en vue d’unegreffe de CSH : la compatibilité HLA entre le donneur et le receveur et le nombre de CNT en fonctiondu poids du receveur. Nous décrirons ici les caractéristiques des USP inscrites sur le registre nationalpar la BSP de Nancy afin de déterminer si ces nouvelles USP inscrites apportent un plus par rapport àtoutes les USP déjà inscrites sur le registre.
... 2D culture of bone-marrow stromal cells is a well-established technique to support in vitro short-term expansion of non-adherent bone-marrow cells. 208 Aiming to improve the necessary expansion of HSCs for clinical applications, engineering strategies have been tested with additional components such as scaffolds, cytokines and supporting stromal cells. PEG-based hydrogels with immobilized SCF and IFN-y enhance the expansion of mouse HSCs and progenitors; however, transplantation assays to confirm the reconstitution ability of expanded HSCs remain to be performed. ...
Adult stem cells are crucial for tissue homeostasis. These cells reside within exclusive locations in tissues, termed niches, which protect adult stem cell fidelity and regulate their many functions through biophysical-, biochemical- and cellular-mediated mechanisms. There is a growing understanding of how these mechanisms and their components contribute towards maintaining stem cell quiescence, self-renewal, expansion and differentiation patterns. In vitro expansion of adult stem cells is a powerful tool for understanding stem cell biology, and for tissue engineering and regenerative medicine applications. However, it is technically challenging, since adult stem cell removal from their native microenvironment has negative repercussions on their sustainability. In this review, we overview specific elements of the biomimetic niche and how recreating such elements can help in vitro propagation of adult stem cells.
... The long-term BM culture assay (Dexter et al., 1977a;Sacchetti et al., 2007) is the principal system in which we can describe the roles of these mesenchymal stromal cells in creating an appropriate hematopoietic microenvironment through the release of cytokines (IL-6, IL-7, IL-8, IL-11, IL-12, IL-15, etc.), growth factors (LIF, G-CSF, GM-CSF, M-CSF, Flt-3, SCF, PDGF, thrombopoietin, etc.), chemokines (SDF-1, RANKL, CCL2, etc.), metalloproteinases (MMP-2, MMP-3, MMP-9, MMP-13, etc.), MMP inhibitors (TIMP-1, TIMP-2, etc.) and extracellular matrix components (fibronectin, collagen I, III and IV, laminin, heparan sulfate, dermatan sulfate, chondroitin sulfate, proteoglycans and hyaluronic acid) (Charbord, 2010;Dexter et al., 1977b;Hofer, 2002;Majumdar et al., 1998). Some factors are involved at various levels of hematopoiesis, at the same time acting as negative or positive regulators of proliferation (TGF-b, MIP-1a, etc.), according to the targeted cells; these factors may also be involved in the control of the proliferation of mesenchymal stromal cells from the BM and proliferation of other tissues (Boiret et al., 2005;De Becker et al., 2007;Kasper et al., 2007;Minguell et al., 2001). ...
... At the cellular level, age-related changes in skeletal health can be attributed largely to declines in both number and function of mesenchymal stromal cells (MSCs) in the bone marrow (D'Ippolito et al. 1999;Kasper et al. 2009;Shen et al. 2011). MSCs are highly proliferative multipotent progenitor cells and have the ability to differentiate into various mesoderm-type cells such as osteoblasts, chondrocytes, adipocytes (Pittenger et al. 1999) and hematopoiesis-supportive stromal cells (Dexter et al. 1977). Hence, they are thought to be the major progenitor cells for intramembranous and endochondral bone formation (He et al. 2013). ...
A focused low-intensity pulsed ultrasound (FLIPUS) was used to investigate the effects of stimulation period, acoustic intensity and donor age on the osteogenic differentiation potential of rat mesenchymal stromal cells (rMSCs). rMSCs from 3- and 12-mo-old female Sprague Drawly rats were isolated from bone marrow and stimulated 20 min/d with either 11.7 or 44.5 mW/cm(2) (spatial average temporal average intensity) for 7 or 14 d. Osteogenic differentiation markers, i.e., Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and degree of matrix calcification were analyzed. On day 7 of stimulation, OCN gene expression was enhanced 1.9-fold in cells from young rats when stimulated with low intensity. The low intensity also led to a 40% decrease in RUNX2 expression on day 7 in aged cells, whereas high intensity enhanced expression of RUNX2 on day 14. FLIPUS treatment with low intensity resulted in a 15% increase in extracellular matrix mineralization in young but not old rMSCs. These differences suggest the necessity of a donor-age related optimization of stimulation parameters.
... Steel mice were cured by surgical spleen implants from a wildtype mouse, whereas white-spotting mice were cured by intravenous injection of wild type marrow cells. Dr. Michael Dexter found that adherent microenvironmental cells from steel mice were able to support the normal differentiation of steel mice marrow cells [110,111], supporting the idea that abnormalities in both the hematopoietic compartment and their microenvironment may lead to abnormal hematologic conditions. We now know that steel mouse is characterized by mutations in the stem cell factor (SCF), which is produced by stromal cells, and white-spotting mice by mutations in c-kit, a receptor present in HPSCs. ...
We review the murine and human microenvironment and hematopoietic stem cell niche in the context of intact bone marrow architecture in man and mouse, both in normal and in myelodysplastic syndrome marrow. We propose that the complexity of the hematopoietic stem cell niche can usefully be approached in the context of its topobiology, and we provide a model that incorporates in vitro and in vivo models as well as in situ findings from intact human marrow to explain the changes seen in myelodysplastic syndrome patients. We highlight the clinical application of the study of the bone marrow microenvironment and its topobiology in myelodysplastic syndromes.
... These different types of niche cells enable hematopoietic stem and progenitor cells (HSPCs) to exert their physiological functions, including migration (either homing or mobilization), self-renewal, differentiation, quiescence and programmed cell death. [4][5][6][7][8] BM mesenchymal stem cells (MSCs) were originally shown to be the self-renewing precursor cells that give rise to bone stromal cells, 9,10 and subsequent studies have documented the critical role of MSCs in the microenvironment for maintaining normal hematopoiesis. For example, Nestin + MSCs co-localize with HSCs in the BM and exhibit increased expression of Cxcl12, Scf, Ang-1 and Vcam-1, which are known regulators of HSCs. ...
Degeneration of normal hematopoietic cells is a shared feature of malignant diseases in the hematopoietic system. Previous studies have shown the exhaustion of hematopoietic progenitor cells (HPCs) in leukemic marrow, whereas hematopoietic stem cells (HSCs) remain functional upon relocation to non-leukemic marrow. However, the underlying cellular mechanisms, especially the specific niche components that are responsible for the degeneration of HPCs, are unknown. In this study, we focused on murine bone mesenchymal stem cells (MSCs) and their supporting function for normal hematopoietic cells in Notch1-induced acute T cell lymphocytic leukemia (T-ALL) mice. We demonstrate that the proliferative capability and differentiation potential of T-ALL MSCs were impaired due to accelerated cellular senescence. RNA-seq analysis revealed significant transcriptional alterations in leukemic MSCs. After co-cultured with the MSCs from T-ALL mice, a specific inhibitory effect on HPCs was defined, whereas in vivo repopulating potential of normal HSCs was not compromised. Furthermore, osteoprotegerin was identified as a cytokine to improve the function of T-ALL MSCs and to enhance normal HPC output via the p38/ERK pathway. Therefore, this study reveals a novel cellular mechanism underlying the inhibition of HPC generation in T-ALL. Leukemic MSCs may serve as a cellular target for improving normal hematopoietic regeneration therapeutically.Leukemia accepted article preview online, 04 August 2015. doi:10.1038/leu.2015.210.
... Since their discovery in the bone marrow by Friedenstein et al. [2], it has been proposed that BMSCs support hematopoiesis through direct and indirect mechanisms. Dexter et al. devised murine long-term cultures in which bone marrow mesenchymal stromal cells could maintain hematopoiesis in vitro [3]. By preserving HSC properties in the bone marrow, BMSCs are therefore key components of the so-called "stem cell niche" [4]. ...
Recent discoveries have significantly expanded our previous knowledge about the role of bone marrow mesenchymal stem cells (BMSCs) in hematopoiesis. BMSCs and their derivatives modulate blood production and immunity at different levels but a prominent role has emerged for BMSCs in the regulation of hematopoietic stem and progenitor cells (HSPCs). Additionally, BMSC-like cells regulate B and T cell lymphopoiesis and also probably myelopoiesis. Furthermore, BMSCs might also exhibit key regulatory properties in non-physiological conditions. BMSCs in extramedullary sites might provide a permissive microenviroment to allow for transient hematopoiesis. BMSCs might be also involved in the manifestation and/or the development of hematopoietic diseases, as steming from their emerging roles in the progression of hematological malignancies. Here we review some key molecular pathways, adhesion molecules and ligand/receptor interactions that mediate the crosstalk between BMSCs and hematopoietic stem cells (HSCs) in health and disease. The development of novel markers to visualize and isolate individual cells will help to dissect the stromal-hematopoietic interplay.
Copyright © 2015. Published by Elsevier B.V.
... The culture system we employed results in formation of multinucleated giant cells responsive to osteotropic hormones capable ofbone resorption and possessing many of the physical and ultrastructural characteristics of osteoclasts. This culture system is dependent on the formation of a marrow adherent cell layer which mimics the marrow microenvironment (28)(29)(30) . However, using a similar technique Dexter and co-workers (28-30) did not report the formation of multinucleated cells in their bone marrow cultures . ...
The predominant cell responsible for bone resorption, the multinucleated osteoclast, has been difficult to study because of inaccessibility. When feline marrow-derived mononuclear cells are established in long-term culture, multinucleated cells form within 48 h, reaching maximum numbers at 16 d. We have observed that these cultured cells have many of the features of osteoclasts. Morphologically, they are multinucleated, contain large numbers of branched mitochondria, have a peripheral cytoplasm lacking organelles (a clear zone), and have extensive cell-surface processes. In addition to these ultrastructural features, the cells contain a tartrate-resistant acid phosphatase, the activity of which is increased by parathyroid hormone (PTH) and inhibited by calcitonin. PTH, prostaglandin E2, and 1,25(OH)2 vitamin D3 increased multinucleated cell formation, while calcitonin inhibited the stimulatory effects of PTH. Time-lapse cinemicrographic and autoradiographic studies indicated that the multinucleated cells formed by fusion of the mononuclear progenitors. The multinucleated cells were phagocytic and stained with nonspecific esterase, consistent with their being derived from immature monocytes. Further, cell populations enriched for multinucleated cells release 45Ca from devitalized bone. Density-gradient centrifugation on Percoll was used to enrich and characterize the mononuclear progenitors of these multinucleated cells. The progenitor cells were found predominantly in Percoll density layers of 1.065 to 1.08 g/ml and were enriched up to 30-fold as compared to unfractionated cells. The bone marrow mononuclear cells that formed the multinucleated cells were initially nonadherent to plastic, stained heavily with nonspecific esterase, and appeared to be immature monocytes histologically. These data indicate that the multinucleated osteoclast-like cells in our cultures are derived from nonadherent monocytic progenitor cells that are responsive to osteotropic hormones. The ability to grow and characterize these cells in vitro should facilitate studies to elucidate the role these cells play in normal and pathologic states of bone resorption.
... There is mounting evidence that cells of the osteoblast lineage, namely osteoprogenitors or mesenchymal stromal cells (MSCs) likely play the most influential supportive roles [21][22][23] together with endothelial cells which have a critical role in HSC maintenance and proliferation in vascular HSC niches [24][25][26]. The first successful efforts to mimic this complex signal milieu, resulting only in transient in vitro HSC maintenance, were reported by Dexter and colleagues [27,28]. In these studies unselected populations of stromal and haematopoietic cells from whole BM were co-cultured. ...
Haematopoietic stem cell (HSC) transplantation is an established cell-based therapy for a number of haematological diseases. To enhance this therapy, there is considerable interest in expanding HSCs in artificial niches prior to transplantation. This study compared murine HSC expansion supported through co-culture on monolayers of either undifferentiated mesenchymal stromal cells (MSCs) or osteoblasts. Sorted Lineage(-) Sca-1(+) c-kit(+) (LSK) haematopoietic stem/progenitor cells (HPC) demonstrated proliferative capacity on both stromal monolayers with the greatest expansion of LSK shown in cultures supported by osteoblast monolayers. After transplantation, both types of bulk-expanded cultures were capable of engrafting and repopulating lethally irradiated primary and secondary murine recipients. LSKs co-cultured on MSCs showed comparable, but not superior, reconstitution ability to that of freshly isolated LSKs. Surprisingly, however, osteoblast co-cultured LSKs showed significantly poorer haematopoietic reconstitution compared to LSKs co-cultured on MSCs, likely due to a delay in short-term reconstitution. We demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSCs without a need for additional haematopoietic growth factors. We also demonstrated that despite apparently superior in vitro performance, co-injection of bulk cultures of osteoblasts and LSKs in vivo was detrimental to recipient survival and should be avoided in translation to clinical practice.
... Given that adherent mesenchymal cells participated in support of hematopoietic cells in vitro (Dexter et al., 1977;Gartner and Kaplan, 1980;Keating et al., 1984), they were tested in vivo, and contradicting data emerged regarding their role in BM transplants based on the hypothesis that they might be transplantable themselves (Bentley et al., 1982;Dexter, 1982;Keating et al., 1982). Studies were highly variable in terms of the pre-transplant conditioning protocols and the heterogeneous and ambiguous nature of the MSC cultures. ...
Mesenchymal stromal cells (MSCs) are heterogeneous and primitive cells discovered first in the bone marrow (BM). They have putative roles in maintaining tissue homeostasis and are increasingly recognized as components of stem cell niches, which are best defined in the blood. The absence of in vivo MSC markers has limited our ability to track their behavior in vivo and draw comparisons with in vitro observations. Here we review the historical background of BM-MSCs, advances made in their prospective isolation, their developmental origin and contribution to maintaining subsets of hematopoietic cells, and how mesenchymal cells contribute to other stem cell niches.
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... However, by 1977 hematologists had already recognized the stromal framework in the bone marrow as the place of origin for these cells. Not surprisingly, hematologists named them mesenchymal stromal cells and used these cells as feeder layers for hematopoietic stem cells (Dexter et al., 1977). In 1991, Caplan and colleagues, focusing on their observations that these cells could be differentiated into bone, cartilage and fat cells given the right culture conditions, named these nonhematopoietic cells from the bone marrow mesenchymal stem cells (Caplan, 1991). ...
Research involving Mesenchymal Multipotent/Stem/Progenitor/Stromal/Marrow cells (MSCs) have translated to clinical trials at an extraordinary pace. By the time of this review, the public clinical trials database (http://clinicaltrials.gov) has 394 clinical trials listed using MSCs for a very wide range of therapeutic applications. Unexpectedly, the explanation for the increase in clinical trials using MSCs does not lie on a well-defined therapeutic mechanism-dramatic results have been demonstrated in a variety of studies involving different animal models of diseases, often describing discrete therapeutic mechanisms exerted by MSCs. This review will focus on recent data suggesting the involvement of hyaluronic acid (HA) in the beneficial effects of MSCs, evaluate the potential of MSC as modulators of HA and the implications of this modulation for disease therapy.
... Attempts to develop in vitro methods that mimic the bone marrow microenvironment in vivo led to the invention of long term bone marrow cultures, represented by the Dexter-type culture for lymphopoiesis (37,38) and the Whitlock-Witte culture for myelopoiesis (39). There has been no such long term culture system available for osteoclastogenesis that is effective in the absence of osteotrophic factors. ...
With advancing age bone marrow is progressively replaced with adipose tissue, accompanied by a concomitant decline in bone
mass and strength. The mechanism underlying the increase in marrow fat and bone destruction remains elusive. We found that
on the way of adipogenic differentiation of marrow stromal cells, receptor activator for NF-κB ligand (Rankl) expression was induced, concomitantly with a down-regulation of osteoprotegerin, which prompted us to hypothesize that cells
at a preadipocyte stage express RANKL. This concept was supported by the findings that the early adipogenic transcription
factors C/EBPβ and C/EBPδ, but not the late factor peroxisome proliferator-activated receptor γ, bind to the Rankl promoter and stimulate Rankl gene transcription. In fact, when cells isolated from the bone marrow of aging mice were analyzed by flow cytometry, we found
that cells expressing the pre-adipocyte marker Pref-1 were RANKL-positive, and the number of these cells was increased with
aging, with concomitant down-regulation of osteoprotegerin, and most importantly, that these RANKL+/Pref-1+ marrow cells were capable of generating osteoclasts from bone marrow macrophages. Thus, the capacity of cells at a pre-adipocyte
stage to express RANKL via C/EBPβ and C/EBPδ and to support osteoclastogenesis may account partly for the co-progression of
fatty marrow and bone destruction with aging.
... Prothymocytes were lost from our long-term cultures before the disappearence of CFU-S was apparent (Tables II,IV). The finding that a high proportion of CFU-S in our cultures were killed with tritiated thymidine is in disagreement with the results of Dexter et al. (1979b), who found that the CFU-5 in their cultures were not cycling 7 days after feeding. This difference might be due to the fact that we used nonselected fetal calf serum supplemented with hydrocortisone in our cultures, whereas they used selected horse serum. ...
In this thesis, the study of growth kinetics of thymocytes in lethally
irradiated mice as a means to characterise the early T-cell progenitors is
described. Normal bone marrow cells were used as the source of hemopoietic cells.
The experiments show that it is possible to obtain information on T-cell differentiation
from a complex experimental set-up in vivo. The quantitative relation
between the thymus regeneration capacity and the CFU-S content of bone marrow
cells was established. K"1netk studies of the regeneration process led to formulation
of the framework for the lil vivo thymus regeneration assay (Chapter 3).
From the study of erythroid and granulocyte-macrophage lineages, it had
been established that at least these lineages start from early progenitor cells that
are immediate successors of the pluripotent cells, but can be distinguished
experimentally from CFU-S (Bol, 1980). However, apart from the cells postulated
by Abramson et al. (1977), no such early progenitor cells are known for the
T-cell 1"1neage.
Our results obtained in thymus repopulation after transplantation of bone
marrow from normal mke did not allow a distinction between the T-cell progenitor
function of prothymocytes and that of the pluripotent stem cell determined as
CFU-5
... The culture system we employed results in formation of multinucleated giant cells responsive to osteotropic hormones capable ofbone resorption and possessing many of the physical and ultrastructural characteristics of osteoclasts. This culture system is dependent on the formation of a marrow adherent cell layer which mimics the marrow microenvironment (28)(29)(30) . However, using a similar technique Dexter and co-workers (28-30) did not report the formation of multinucleated cells in their bone marrow cultures . ...
The predominant cellresponsibleforbone resorption,the multinucleatedosteo- clast,has been difficultto study because of inaccessibility .When felinemarrow-derived mononuclear cellsareestablishedinlong-termculture,multinucleatedcellsform within48 h, reachingmaximum numbers at16 d.We have observed thattheseculturedcellshave many ofthefeaturesofosteoclasts.Morphologically,theyaremultinucleated,containlargenumbers ofbranched mitochondria,have a peripheralcytoplasm lackingorganelles(aclearzone),and have extensivecell-surfaceprocesses.Inadditionto these ultrastructural features,the cells containa tartrate-resistant acid phosphatase,the activityofwhich isincreasedby parathyroid hormone (PTH) and inhibitedby calcitonin.PTH, prostaglandinE2,and 1,25(OH)2vitaminD 3 increasedmultinucleatedcellformation,while calcitonininhibitedthe stimulatoryeffectsof PTH .Time-lapse cinemicrographicand autoradiographicstudiesindicatedthatthe multinu- cleatedcellsformed by fusionofthe mononuclear progenitors.The multinucleatedcellswere phagocyticand stainedwith nonspecificesterase,consistentwith theirbeing derived from immature monocytes .Further,cellpopulationsenriched formultinucleatedcellsrelease"Ca from devitalizedbone . Density-gradientcentrifugationon Percollwas used to enrich and characterizethe mononuclear progenitorsofthese multinucleatedcells.The progenitorcells were found predominantly inPercolldensitylayersof 1.065to 1.08g/mland were enriched up to 30-foldas compared tounfractionatedcells.The bone marrow mononuclear cellsthat formed the multinucleatedcellswere initially nonadherent to plastic,stainedheavilywith nonspecificesterase,and appeared to be immature monocytes histologically .These data indicatethatthe multinucleatedosteoclast-like cellsinour culturesaredevived from nonad- herentmonocytic progenitorcellsthatareresponsiveto osteotropichormones .The abilityto grow and characterizethese cellsinvitroshould facilitate studiesto elucidatethe rolethese cellsplayinnormal and pathologicstatesofbone resorption.
Significance
Recapitulating human blood cancers in vitro remains challenging due to the limitations of current models to culture patient-derived malignant hematopoietic stem and progenitor cells in entirely human bone marrow microenvironments. We demonstrate that progenitor cells from patients with acute myeloid leukemia and myeloproliferative neoplasms can be cultured for at least 3 wk in fully human cell-based three-dimensional osteoblastic niches engineered in perfusion bioreactors, while exhibiting key features found in native bone marrow. Furthermore, the system can be customized to include a human vascular component within the engineered stromal microenvironment, which enables investigation of human leukemogenesis under designed settings. This platform can be used to test the effectiveness of chemotherapy compounds, toward application in patient-personalized medicine.
Although the presence of cell adhesion molecules on primitive haemopoietic cells are known and well characterised, adhesion pathways mediated by many of these CAMs remain a matter of intense investigation. This thesis explores the adhesive interactions of haemopoietic stem cells (HSC) and progenitors with ECM ligands and stroma with particular emphasis on characterising the CD44:HA adhesion pathways. The myeloid cell line, KGla, expressing the CD34 molecule demonstrated significant binding to stroma and less so to HA, this was mediated by CD44. Long-term culture-initiating cell (LTC-IC) and plastic adherent (PA) cells (defined as stem cells) were quantified, in adult BM and peripheral blood (PB), using either limiting dilution assay (LDA) or single dilutions and both showed frequencies similar to previous studies. Adhesion assays using BM MNC and PB CD34+ cells on ligand-coated surfaces, showed that the frequencies of stem cells (either LTC-IC or PA cells), in the nonadherent fraction were less compared with those in the original cell suspension. These data suggest that there was 2-3 fold enrichment of of the two stem cell population by HA.The role of CD44 in the adherence of the stem cells to the ligands was investigated by including anti-CD44 moAb in adhesion assays and in two experiments both stem cells populations showed partial inhibition of adhesion to HA, compared with control antibody.Adhesion of these stem cell populations to fibronectin were also studied. Similar to HA, the frequencies in the two stem cells were higher in the original than in the nonadherent cell suspension suggesting enrichement by FN. The adhesion was blocked by anti-integrin pi antibody but not by anti-CD44 antibody. CFU-GM progeny assayed from PB CD34+ cells were shown to adhere to HA, furthermore this adhesion was partially inhibited by anti-CD44 antibody. Similarly theadhesion of CFU-C progeny assayed from UCB cell fractions was inhibited by the antibody.These findings suggest that the majority of stem cells in BM, PB and progeny in UCB cell populations are adherent to HA and that this is dependent, at least in part, on CD44.
The ultimate goal of separating the constituent cell populations in bone marrow is the identification of the multiple phenotypes present within the heterogeneous marrow community, potentially for use in clinical therapies. These cell populations are small in number and show diversity in their origins and differentiation capability. This presents a major challenge for the methodologies that have been developed for isolation and separation. This chapter outlines the markers that have been identified for the isolation of different fractions from within a bone marrow sample, including mesenchymal stem cells. Routine approaches for large-scale identification have been put into practice for isolating stromal and hematopoietic populations, while other markers for many of the subpopulations are still being defined. Technologies that have been developed for the isolation of stem cell populations within bone marrow ranging from magnetic to optical to microfluidic techniques are described. Current research includes developing single-cell technologies for isolating and defining low numbers of stem cell populations that can form cloned stock populations for allogeneic therapies. These technologies form an important basis for ultimately bringing cell therapies to the clinic for treatment of a variety of diseases.
Hemonectin, a component of bone marrow extracellular matrix, is a lineage- and organ-specific attachment molecule for cells of the granulocytic lineage. We hypothesized that hemonectin is an important marker of fetal granulopoiesis that is developmentally regulated during the ontogeny of the hematopoietic system. Murine hematopoiesis originates in the yolk sac and subsequently appears in the liver, spleen, and bone marrow. Using an affinity-purified polyclonal antibody to purified hemonectin as a probe of developing hematopoietic organs, we observe that hemonectin is coordinately expressed at developmental stages of the mouse in those tissues that are supporting hematopoiesis. Multiparameter flow cytometric analysis reveals that approximately 7% of fetal liver cells express hemonectin by day 13 of gestation, and that 32% of the cells are positive by day 19. Additionally, restricted hemonectin expression is noted in other tissues (cartilage, skin, developing bone, and capillary endothelial cells), suggesting that this molecule subserves other developmental functions and/or belongs to a previously unrecognized family of molecules.
Stem cells have a proven record of safety and efficacy over the past 50 years of research. Regenerative therapy continues to be an extremely active field of basic science and translational research with significant promise in treatment of previously untreatable pathophysiology. The fast development in epigenetics, genetic engineering technologies, and 3D printing is opening new frontiers in advancing the utility of stem cells, and minimally invasive technology provides a platform for clinical application with promising results. This chapter outlines the basic characterizations of stem cells, reviews their history and supporting evidence, and explores some of the clinical applications currently under investigation.
In contrast to non-mammalian vertebrates, mammals and humans have limited innate capacity for the self-regeneration of tissues and organs owing to differences in genetics, development, immune systems and tissue complexity. Endogenous stem cells are tissue-specific adult stem cells with the capacity to self-renew and differentiate into specific cell types. Therefore, endogenous stem cells are being explored for the regeneration of tissues in situ and in vivo. Stem cells reside in specific niches in the body, and stem cell activation depends on progressive changes in the niche. Niches are specific and instructive microenvironments that can be recreated using biomaterial-based scaffolds. Such scaffolds can be fabricated into a variety of shapes and formulations, and they can be functionalized with biochemical and biophysical cues to guide stem cell fate and migration. In this Review, we discuss important differences in the self-regeneration abilities of non-mammalian vertebrates and mammals, including humans, and investigate adult stem cell populations and their niches involved in tissue repair and regeneration. We highlight natural and synthetic biomaterials and their potential for improving applications of endogenous stem cells and examine the role of interspecies chimaeras in regenerative medicine.
Purpose of review:
Hematopoietic stem cells (HSCs) predominantly reside either in direct contact or in close proximity to the vascular endothelium throughout their lifespan. From the moment of HSC embryonic specification from hemogenic endothelium, endothelial cells (ECs) act as a critical cellular-hub that regulates a vast repertoire of biological processes crucial for HSC maintenance throughout its lifespan. In this review, we will discuss recent findings in endothelial niche-mediated regulation of HSC function during development, aging and regenerative conditions.
Recent findings:
Studies employing genetic vascular models have unequivocally confirmed that ECs provide the essential instructive cues for HSC emergence during embryonic development as well as adult HSC maintenance during homeostasis and regeneration. Aging of ECs may impair their ability to maintain HSC function contributing to the development of aging-associated hematopoietic deficiencies. These findings have opened up new avenues to explore the therapeutic application of ECs. ECs can be adapted to serve as an instructive platform to expand bona fide HSCs and also utilized as a cellular therapy to promote regeneration of the hematopoietic system following myelosuppressive and myeloablative injuries.
Summary:
ECs provide a fertile niche for maintenance of functional HSCs throughout their lifecycle. An improved understanding of the EC-HSC cross-talk will pave the way for development of EC-directed strategies for improving HSC function during aging.
Despite many advances in blood banking technology, our ability to support patients with blood products remains severely limited because they can only be obtained by harvesting mature cells from the peripheral blood of individual donors. The supply of all cell lines is threatened by occasional shortages and the unavailability of unusual antigen types. White blood cell transfusions have the added problems of inadequate yields, despite advances in leukapheresis, and poor granulocyte function after manipulation, so that white cell transfusions remain of marginal clinical utility [1]. Availability is further limited by the lack of storage techniques for platelets and white cells. Disease transmission remains a problem with conventional techniques [2]. The recent adoption of HTLV-III antibody screening should lessen the transmission of AIDS, but non-A non-B hepatitis remains a frequent complication of transfusion, as is CMV in the immunosuppressed [3] and malaria in Third World countries
The initial attempts to grow bone marrow cells in semi-solid or liquid medium promoted only the terminal differentiation of hematopoietic stem cells in the absence of self-renewal, with cultures becoming hematopoietically unproductive after 1–2 weeks of culture. Subsequent work in the murine system by Dexter and co-workers demonstrated that the sustained growth of hematopoietic cells in liquid culture was possible if these cells were plated onto a pre-established monolayer of bone marrow stromal cells3. These cells synthesize the matrix necessary to support hematopoiesis and contribute trophic/regulatory factors to the hematopoietic microenvironment. Inclusive of this group are fibroblasts, adipocytes reticular adventitial cells, macrophages, and endothelia4. The Dexter long-term bone marrow culture (LTBMC) system sustains the self renewal of murine pluripotential stem cells (CFU-S) although the lineage restricted progenitors become predominantly myeloid in character after the first several weeks of culture3,5. This system can be modulated to favor the growth of lymphoid cells6,7 or erythroid progenitors8, but single cultures do not produce substantial numbers of each hematologic cell type concurrently.
Blood stem cell transplantation aims to regenerate the entire hematopoietic system. Until recently, a significant proportion of patients in need of a hematopoietic cell transplant could not undergo transplantation due to the lack of a suitable matched related or unrelated adult donor. With the advent of umbilical cord blood harvesting and processing technology, the majority of patients can now receive a suitable transplant. This chapter will review current cord blood engineering strategies with a particular focus on novel concepts to facilitate blood stem cell engraftment and hematopoietic reconstitution. Because blood stem cell transplantation serves as the model system for many novel regenerative stem cell therapy strategies, the concepts described are considered to be applicable to nonhematopoietic stem cell therapy as well. Groundbreaking laboratory-based cell processing technologies and strategies to improve engraftment will be discussed.
A number of studies have investigated the properties of murine macrophages grown from bone marrow or peritoneal precursors in short-term culture, i.e. less than two weeks (Table 1). A source of myeloid or macrophage colony-stimulating factor (CSF) is essential for this growth (Stanley et al. 1976). Thus, using a source of predominately macrophage CSF, Watson et al. (1974) showed that liquid cultures would yield approximately 106 marrow cells) in seven days, over 95% with macrophage properties of morphology, adherence, carbonyl iron phagocytosis, neutral red pinocytosis, binding of glutaraldehyde-treated and antibody-sensitized RBC, and lysis of the latter. Marrow-derived macrophages have been an ideal subject for studies on in vitro activation of tumor cytotoxicity, since it is very difficult to maintain animals in the constant or germ-free evironment required for harvesting unstimulated control macrophages (Meerpohl et al. 1976).
In this chapter we would like to review evidence supporting the concept that there is extensive heterogeneity of mammalian mononuclear phagocytes. Given the limits of this chapter, we will summarize data supporting this notion rather than provide an in-depth review of particular characteristics and functions of these cells. Therefore, the main subjects will be the mononuclear phagocyte system as a whole and the heterogeneous features of the various members of the system expressed both phenotypically and functionally. Finally, we will discuss various models that may explain how mononuclear phagocyte heterogeneity is generated (see also Chapter 3, this volume).
Hematopoietic stem cells (HSC) are associated ex vivo with a cell subset actively rejecting lipocationic dye such as rhodamine 123 (Rho) in the extracellular medium via the multi-drug resistance gene product. It is however not known whether this association remains after culture. We therefore evaluated whether cultures derived from CD34+ human cord blood (CB) cells contained dye-excluding (Rho-low) cells enriched in early HSC. Our investigations showed that after short-term culture with FLT3-ligand, thrombopoietin and stem cell factor, Rho-low cell were still detectable, but with a lower frequency than that of uncultured cells. Rho-low cell frequency varied depending on the stimulus used in vitro, and these variations correlated with that of long term-culture initiating cell (LTC-IC) frequencies (R=0.619, p<0.04), whereas no correlation could be established with late HSC (CFU) frequency. Rho-low cells sorted after 6 days of culture contained 2.8-fold more secondary CFU and 1.8 more LTC-IC compared to Rho-high cells. Though these data suggest that dye exclusion assays could be used as a relevant alternative compared to other in vitro assays to identify immature HSC after culture, it remains to be determined whether Rho-low cells derived from cultures retain clinically relevant characteristics such as the ability to reconstitute a lethally conditioned host. In this light, we further discuss below the limitations of some in vitro and in vivo hematopoietic assays, and their relevance as tools to identify HSC that are able rescue conditioned patients.
Mammalian bone marrow is a complex organ responsible for a variety of critical homeostatic functions, including haematopoiesis. To achieve these functions, it has evolved to provide an environment for the habitation of stem cells—the bone marrow ‘stem cell niche’. In addition to haematopoetic stem cells, which are responsible for generating all cell lineages of the mammalian blood, recent evidence suggests that this same niche may provide an environment for the putative skeletal stem cell, responsible for forming the connective tissues of the skeleton—bone, cartilage and fat. In this chapter, we review recent research on the importance of skeletal stem cells and bone marrow stromal cells and their spatial localisation within the bone marrow. We discuss their role in providing a supportive microenvironment for the maintenance of haematopoetic stem cells, and some of their key molecular interactions via cell surface ligands, secreted growth factors, extracellular matrix and other physiocochemical means. We also discuss some of the pathologies that might arise from dysregulation of the niche, particularly with regard to ageing. Finally, we review recent attempts to recreate the bone marrow microenvironment in vitro.
Blood cell production is maintained throughout life by hematopoietic stem cells (HSC), which reside in specific areas of the bone marrow (BM) referred to as niches. These niches regulate the self-renewal, proliferation, and migration of HSC and also integrate signals from the periphery to respond to the hematopoietic demand. In the last decade, several putative cellular components of the HSC niche have been identified. Here, we briefly review current knowledge on different putative niche cells and their regulation.
Haematopoietic stem cell (HSC) transplantation is an established cell-based therapy for a number of haematological malignancies and immunodeficiency diseases. However, the limited number of HSC from umbilical cord blood (UCB) limits the efficacy of transplants from this source. This limitation could be overcome by expanding the HSC population prior to transplantation. Although such processes have shown little success to date, it is thought that self-renewal of HSC in vitro may be possible through replication of the environmental cues found in the bone marrow (BM) stem cell niche. It is thought that non-haematopoietic cell types residing in the putative HSC niche could provide these cues. Mesenchymal stem/stromal cells (MSC) are one such cell type found in the BM niche that provide these cues. Thus, this review will explore how MSC have been used in the ex vivo expansion of HSC.
Sulfated glycosaminoglycans (GAGs) are distributed in consistent and distinctive patterns between the cell surface and the growth medium of haemopoietically active long-term bone marrow cultures. Heparan sulfate is the main cell surface component and chondroitin sulfate is the major sulfated species in the medium. When the cultures are supplemented with beta-D-xylosides a significant increase in chondroitin sulfate synthesis is observed but no stimulation of heparan sulfate synthesis occurs. The chondroitin sulfate accumulates in the culture medium in beta-D-xyloside-treated cultures but the composition of sulfated GAGs in cell-surface derived material is unaffected. beta-D-xylosides also stimulate the production of haemopoietic cells without any apparent alteration in the adherent stromal cells of the marrow cultures. Equivalent increases are obtained in cells at all stages of development so that a fivefold increase in pluripotent stem cells (CFU-S) is matched by fivefold increase in the granulocyte-macrophage progenitors (GM-CFC) and in mature granulocytes. The stimulation persists for many weeks in beta-D-xyloside-treated cultures. These results indicate that the sulfated GAGs may play an important role in the regulation of haemopoiesis.
Adult stem cells persist lifelong in the organism, where they are responsible for tissue homeostasis and repair. It is commonly assumed that their maintenance and function are facilitated in local environments called "stem cell niches." Although there is convincing evidence that a variety of niche components determine stem cell fate, the regulatory details of stem cell-niche interactions are widely unknown. To pave the way for a substantiated discussion of these interactions, we first focus on the stem cells themselves and describe the stem cell defining criteria and their implications. The fate of the cells that fulfill these criteria is regulated by a broad spectrum of factors and regulatory mechanisms. A summary of established components and their action is given exemplary for the hematopoietic system. The complexity resulting from the interplay of various cell types, signaling molecules, and extracellular structures can be boiled down to important key features as exemplified by the presented model of hematopoietic stem cell organization. Although neglecting many details, we show that this and similar models have the power to yield intriguing results as proven by the agreement of the presented model with experimental data and the predictions derived from model simulations. Finally, we will discuss the paradigm of systems biology and give a summary of the techniques that promise to unveil further details of the organization principles of stem cell niches at different levels. The synergistic effect of the described techniques together with the integration of their results into a unified model that allows quantitative evaluation and predictions may lead to a better and more systematic understanding of the most relevant niche elements and their interactions.
Current in vitro hematopoiesis models fail to demonstrate the cellular diversity and complex functions of living bone marrow; hence, most translational studies relevant to the hematologic system are conducted in live animals. Here we describe a method for fabricating 'bone marrow-on-a-chip' that permits culture of living marrow with a functional hematopoietic niche in vitro by first engineering new bone in vivo, removing it whole and perfusing it with culture medium in a microfluidic device. The engineered bone marrow (eBM) retains hematopoietic stem and progenitor cells in normal in vivo-like proportions for at least 1 week in culture. eBM models organ-level marrow toxicity responses and protective effects of radiation countermeasure drugs, whereas conventional bone marrow culture methods do not. This biomimetic microdevice offers a new approach for analysis of drug responses and toxicities in bone marrow as well as for study of hematopoiesis and hematologic diseases in vitro.
The induction of pluripotency in somatic cells is widely considered to be a major breakthrough in regenerative medicine because this approach provides the basis for individualized stem cell–based therapies. Initial reports demonstrated the generation of induced pluripotent iPS cells (iPSCs) from fibroblasts. Since then, pluripotency has been induced in a variety of cell lineages suggesting that the majority of somatic cell types, if not all cells, can be reprogrammed. For broad clinical application, easily accessible cell sources that allow for efficient derivation of patient-specific iPSCs of high biological quality are required. Clearly, blood represents one of the most easily accessible cell sources, and techniques for the induction of pluripotent stem cells from different blood cell types are summarized in this chapter.
The existence of a bone marrow (BM) niche-the location in which hematopoietic stem cells (HSCs) reside-was proposed more than 30 years ago. Recent data suggest that the interaction of HSCs with cellular and extracellular components within the BM is critical for HSC regulation. The tracking of immunofluorescently labeled, prospectively isolated HSCs to and within the BM cavity allows the assessment of the regulatory processes involved in (1) homing, which involves transendothelial migration into the BM; (2) lodgment, including transmarrow migration through the extravascular space; and (3) BM reconstitution. Together, such analyses provide a better understanding of the cellular and extracellular components involved in the regulation of HSC quiescence and differentiation. Homing and lodgment of transplanted HSCs, the first critical steps in engraftment, involve multiple interactions between HSCs and the BM microenvironment. Herein, we describe a refined method of analyzing homing efficiency and spatial distribution of HSCs harvested from endosteal and/or central BM regions; we also review alternate methods. Using these techniques, microenvironment modifications within the recipient or surface protein-expression modifications on donor HSCs in animal models provide insights into components influencing the homing, lodgment, and engraftment processes.
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