An Ectopic Stromal Implant Model for Hematopoietic Reconstitution and in Vivo Evaluation of Bone Marrow Niches

Abstract

In adults, hematopoiesis takes places in the bone marrow, where specialized niches containing mesenchymal nonhematopoietic cells (stroma) harbor the hematopoietic stem cell (HSC). These niches are responsible and essential for the maintenance of HSCs. Attempts to expand HSCs fail to keep the general properties of stem cells, which depend on several niche components difficult to reproduce in in vitro culture systems. Here, we describe a methodology for in vivo study of hematopoietic stroma. We use stroma-loaded macroporous microcarriers implanted in the subcutaneous tissue of experimental animals and show that the ectopic stroma implant (ESI) is able to support hematopoiesis. Moreover, lethally irradiated mice can be rescued by ESI preloaded with HSCs, showing that they function as an ectopic bone marrow. ESI is also shown as a good system to study the role of different niche components. As an example, we used stromas lacking connexin 43 (Cx43) and confirm the importance of this molecule in the maintenance of the HSC niche in vivo. We believe ESI can work as an ectopic bone marrow allowing in vivo testing of different niches components and opening new avenues for the treatment of a variety of hematologic conditions particularly when stromal cell defects are the main cause of disease.

Multifaceted Roles of Connexin 43 in Stem Cell Niches

“…Although Cx43-deficient (Cx43 −/− ) mice die shortly after birth due to heart malformations [ 86 ], precluding postnatal hematopoietic studies, embryonic Cx43 −/− mice exhibit reduced fetal liver HSC and progenitor cells. Cx43 deficiency in stromal cells, specifically, results in a reduction of functional HSC and progenitor cells in the fetal liver and impairs the growth and differentiation of bone marrow HSC, suggesting that Cx43 acts as critical regulator of hematopoiesis [ 87 , 88 ]. Furthermore, Cx43 was shown to be critical for injury-induced hematopoietic regeneration in adults [ 89 ].…”

Decline of FOXN1 gene expression in human thymus correlates with age: possible epigenetic regulation

“…Cells were cultured in 10 % fetal calf serum (Cultilab, São Paulo, Brazil) supplemented RPMI 1640 medium (Mediatech, Virginia, USA) at 37 °C in a 5 % CO 2 atmosphere. Alternatively, these cells were cultured in three-dimensional cell culture system using macroporous cellulose microcarriers (Cytopore™, Asaki Kasei Medical Co., GE Healthcare, Japan) as described previously [ 63 ] with some modifications. Briefly, 100 μL of phosphate-buffered saline (PBS)-hydrated macroporous cellulose microcarriers were colonized with cells in a volume of the 2x10 5 cells under nitrocellulose membrane from Transwell™ inserts (Costar, Corning Incorporated, USA) and cultured for 4 days with complete medium at 37 °C in a 5 % CO 2 atmosphere.…”

Moving Receptor Redirected Adoptive Cell Therapy Toward Fine Tuning of Antitumor Responses

“…For stem cell-targeted rdACT, in vitro and in vivo models that enable the selective evaluation of stem cell-based tumor cell renewal are valuable tools. Such models are available for normal [98] and leukemic [99] hematopoiesis in vitro and in vivo [100,101], and for some models of xenografted tumors [93,100,102].…”