An in vivo model to study and manipulate the hematopoietic stem cell niche
ABSTRACT Because the microenvironment that supports hematopoietic stem cell (HSC) proliferation and differentiation is not fully understood, we adapted a heterotopic bone formation model as a new approach for studying the HSC microenvironment in vivo. Endogenous HSCs homed to tissue-engineered ossicles and individually sorted HSCs from ossicles were able to reconstitute lethally irradiated mice. To further explore this model as a system to study the stem cell niche, ossicles were established with or without anabolic parathyroid hormone (PTH) treatment during the 4-week course of bone development. Histology and micro-computed tomography showed higher bone area-to-total area ratios, thicker cortical bone and trabecular bone, significantly higher bone mineral density and bone volume fraction in PTH-treated groups than in controls. By an in vivo competitive long-term reconstitution assay, HSC frequency in the ossicle marrow was 3 times greater in PTH groups than in controls. When whole bone marrow cells were directly injected into the ossicles after lethal irradiation, the PTH-treated groups showed an enhanced reconstitution rate compared with controls. These findings suggest the residence of HSCs in heterotopic bone marrow and support the future use of this ossicle model in elucidating the composition and regulation of the HSC niche.
- SourceAvailable from: Boris Michael Holzapfel
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- "Moreover, some of these studies show none or only small amounts of mineralized tissue formation e not a fully regenerated organ bone e and often the newly formed bone and marrow compartment is interspersed with large volumes of scaffold material, which interferes with the development of a coherent physiological tissue network  . Importantly, most current models investigating hematopoietic niche physiology fail to recapitulate the processes seen in humans as they analyse the behaviour and function of murine e and not human e HSCs within their niches   . "
ABSTRACT: Advances in tissue-engineering have resulted in a versatile tool-box to specifically design a tailored microenvironment for hematopoietic stem cells (HSCs) in order to study diseases that develop within this setting. However, most current in vivo models fail to recapitulate the biological processes seen in humans. Here we describe a highly reproducible method to engineer humanized bone constructs that are able to recapitulate the morphological features and biological functions of the HSC niches. Ectopic implantation of biodegradable composite scaffolds cultured for 4 weeks with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone organ including a large number of human mesenchymal cells which were shown to be metabolically active and capable of establishing a humanized microenvironment supportive of the homing and maintenance of human HSCs. A syngeneic mouse-to-mouse transplantation assay was used to prove the functionality of the tissue-engineered ossicles. We predict that the ability to tissue engineer a morphologically intact and functional large-volume bone organ with a humanized bone marrow compartment will help to further elucidate physiological or pathological interactions between human HSCs and their native niches. Copyright © 2015. Published by Elsevier Ltd.Biomaterials 08/2015; 61:103 - 114. DOI:10.1016/j.biomaterials.2015.04.057 · 8.56 Impact Factor
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ABSTRACT: A prospective in vivo assay was used to identify cells with potential for multiple lineage differentiation. With this assay, it was first determined that the 5-fluorouracil resistant cells capable of osseous tissue formation in vivo also migrated toward stromal derived factor-1 (SDF-1) in vitro. In parallel, an isolation method based on fluorescence-activated cell sorting was employed to identify a very small cell embryonic-like Lin-/Sca-1+CD45- cell that with as few as 500 cells was capable of forming bone-like structures in vivo. Differential marrow fractionation studies determined that the majority of the Lin-Sca-1+CD45- cells reside in the subendosteal regions of marrow. To determine whether these cells were capable of differentiating into multiple lineages, stromal cells harvested from Col2.3 Delta TK mice were implanted with a gelatin sponge into SCID mice to generate thymidine kinase sensitive ossicles. At 1.5 months, 2,000 green fluorescent protein (GFP)+ Lin-Sca-1+CD45- cells were injected into the ossicles. At harvest, colocalization of GFP-expressing cells with antibodies to the osteoblast-specific marker Runx-2 and the adipocyte marker PPAP gamma were observed. Based on the ability of the noncultured cells to differentiate into multiple mesenchymal lineages in vivo and the ability to generate osseous tissues at low density, we propose that this population fulfills many of the characteristics of mesenchymal stem cells.Stem cells and development 05/2010; 19(10):1557-70. DOI:10.1089/scd.2009.0445 · 3.73 Impact Factor
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ABSTRACT: Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide an accessible, genetically tractable, and homogenous starting cell population to efficiently study human blood cell development. These cell populations provide platforms to develop new cell-based therapies to treat both malignant and nonmalignant hematological diseases. Our group previously demonstrated the ability of hESC-derived hematopoietic precursors to produce functional natural killer (NK) cells as well as an explanation of the underlying mechanism responsible for the inefficient development of T and B cells from hESCs. hESCs and iPSCs, which can be engineered reliably in vitro, provide an important new model system to study human lymphocyte development and produce enhanced cell-based therapies with the potential to serve as a "universal" source of antitumor lymphocytes. This review will focus on the application of hESC-derived NK cells with currently used and novel therapeutics for clinical trials, barriers to translation, and future applications through genetic engineering approaches.09/2010; 156(3):147-54. DOI:10.1016/j.trsl.2010.07.008