L’hématopoïèse est un processus finement régulé pour permettre sa pérennité et son adaptation aux contraintes physiologiques et pathologiques. Ce potentiel repose en grande partie sur les capacités de quiescence, auto-renouvellement, division asymétrique et multipotence des cellules souches hématopoïétiques (CSH). Les CSH et progéniteurs hématopoïétiques (CSPH) sont principalement régulés de façon extrinsèque au sein des niches hématopoïétiques médullaires et cette régulation fait intervenir, des contacts intercellulaires et des facteurs diffusibles. Le phénotype « side-population » (SP), secondaire à l’efflux actif d’un colorant fluorescent (Hoechst 33342) par des pompes de type multidrugresistance, est une caractéristique des cellules souches de la plupart des tissus. Au sein de l’hématopoïèse, le phénotype SP est un excellent moyen pour identifier les CSH murines et est associé à leur quiescence et à leur adhésion à la niche endostéale, mais sa valeur comme marqueur des CSH est plus discutée chez l’homme. Les cellules SP, de par leur nature, sont également étudiées en oncologie, et sont associées aux cellules tumorales les plus résistantes et les plus tumorogènes. La compréhension des mécanismes régulant la fonctionnalité SP devrait permettre d’ouvrir des pistes en physiologie quand à la compréhension de la régulation des CSPH par les niches mésenchymateuses et en pathologie pour cibler les mécanismes de chimiorésistance.Dans ce travail nous montrons pour la première fois chez l’homme que l’acquisition du phénotype SP est un phénomène dynamique et versatile sous le contrôle du stroma médullaire. Le stroma médullaire est en effet capable de maintenir le phénotype SP de CSPH médullaires et d’induire le phénotype SP de CSPH circulants. L’acquisition du phénotype SP par les cellules circulantes nécessite à la fois un « nichage » au sein du stroma et des facteurs diffusibles. Les cellules circulantes capables d’acquérir le phénotype SP contiennent des CSPH au regard de (i) leur expression du CD34, (ii) leur richesse en cellules quiescentes, (iii) leur capacité clonogénique et proliférative en cultures secondaires, (iv) leur expression des gènes de « nichage » et de « souchitude », (v) leur capacité de migration en réponse à un gradient de CXCL12, (vi) leur activité LT-SRC in vivo. De plus nous avons mis en évidence, au sein de ces CSPH SP+CD34+ révélés par le stroma médullaire, une sous-population CD44-/faible qui pourrait contenir les cellules plus immatures en raison de sa quiescence et de l’intensité de son efflux du Hoechst 33342. Les études mécanistiques montrent que l’acquisition du phénotype SP par les cellules circulantes est sous la dépendance de l’intégrine VLA-4 et du CD44. La transduction du signal implique des protéines G et la famille des Src-kinases. Nous montrons également que le stroma médullaire peut induire/maintenir/amplifier la fonctionnalité SP de blastes circulants de leucémie aigüe myéloblastique de façon ß1-intégrine dépendante et que cette fonctionnalité est associée à une capacité d’efflux de Mitoxantrone. Ce mécanisme de modulation de l’activité d’ABC-transporteurs par l’adhésion au stroma correspond à un mécanisme encore jamais décrit de CAM-DR. / Hematopoiesis is a finely tuned process to allow its long-term efficiency and its adaptation to various physiological and pathological stresses. Hematopoietic stem cell (HSC) is the keystone of hematopoiesis through its multipotency, quiescence, asymmetrical division and self-renewing properties. HSC bone marrow (BM) niches mainly regulate hematopoietic stem and progenitor cells (HSPC) through intercellular contacts and diffusible factors. Side-population (SP) cells are characterized by their capability to actively efflux Hoechst 33342 dye through multidrug resistance-like pumps. SP phenotype is a characteristic of stem cells in many tissues and especially, it is a stringent criterion to purify murine HSCs. In mice, this phenotype has been demonstrated to be related to quiescence and resistance to drugs/environmental stresses and to be controlled by endosteal niche adhesion. SP cells are also studied in oncology and are associated to chemo-resistance and tumor initiating capacity. At steady state, SP cells are mainly present in the BM and are mostly absent from the circulation except in stress conditions, raising the hypothesis of the versatility of the SP functionality. Therefore, studying SP phenotype regulation is of importance to understand how BM niches regulate HSPC and how to interfere with cancer cells chemo-resistance.In this work, we demonstrate for the first time and in human that SP phenotype acquisition is a dynamic phenomenon under control of stromal BM cells. Stromal cells from healthy donors maintain SP phenotype of BM HSPC and promote SP phenotype acquisition in circulating ones. SP phenotype promotion depends of stroma nesting and of diffusible factors secretion. This stroma-induced circulating SP cell fraction contains HSPC, as ascertained by (i) CD34 expression, (ii) proportion of cells in G0, (iii) clonogenic and proliferative potential, (iv) nesting and “stemness” gene expression, (v) CXCL12-related migration capability and (vi) LT-SRC activity. Moreover, we describe an SP+CD34+CD44-/low sub-population that could contain most immature HSPCs with regards to their quiescence and Hoechst efflux intensity. Mechanistic studies show that the stoma-mediated SP promoting effect is VLA-4/4ß1-integrin and CD44 dependent, and implicate G-protein and Src-kinase pathways. We also demonstrate that BM stroma from healthy donors can induce/maintain/amplify in a ß1-integrin dependent manner an SP sub-population with mitoxantrone efflux capability in blast cells from acute myeloid leukemia. The existence of a similar mechanism in circulating leukemic blasts suggests the possibility to interfere with the chemo-resistant phenotype of blast cells through integrin/CD44 axis blockade.

Background: Multipotent mesenchymal stromal cells (MSC) can be recovered from a variety of tissues in the body. Yet, their functional properties were shown to vary depending on tissue origin. While MSC have emerged as a favoured cell type for tendon regenerative therapies, very little is known about the influence of the MSC source on their properties relevant to tendon regeneration. The aim of this study was to assess and compare the expression of tendon extracellular matrix proteins and tendon differentiation markers in MSC derived from different sources as well as in native tendon tissue. MSC isolated from equine bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood and tendon tissue were characterized and then subjected to mRNA analysis by real-time polymerase chain reaction. Results: MSC derived from adipose tissue displayed the highest expression of collagen 1A2, collagen 3A1 and decorin compared to MSC from all other sources and native tendon tissue (p < 0.01). Tenascin-C and scleraxis expressions were highest in MSC derived from cord blood compared to MSC derived from other sources, though both tenascin-C and scleraxis were expressed at significantly lower levels in all MSC compared to native tendon tissue (p < 0.01). Conclusions: These findings demonstrate that the MSC source impacts the cell properties relevant to tendon regeneration. Adipose derived MSC might be superior regarding their potential to positively influence tendon matrix reorganization.

Dogs are useful preclinical models for the translation of cell transplantation therapies from the bench to the bedside. In order for canine models to be utilized for stem cell transplantation research, it is necessary to advance discoveries in the fields of canine stem cell biology and transplantation. The use of side population hematopoietic stem cells (HSCs) has garnered much interest for the purification of mouse HSCs and has been translated to several other species, including human. In order to assess if this method of purification of HSCs could be useful for stem cell therapies in humans, safety and efficacy studies in a large animal model, such as the dog would be required. With this objective in mind, we isolated canine bone marrow-derived side population (SP) stem cells and assessed their multilineage differentiation in vitro and engraftment potential in vivo. Utilizing a pregating strategy to enrich for small, agranular SP cells we were able to enrich for blast cells, expressing the ABCG2 transmembrane pump known to be associated with murine and human SP cells. Canine SP cells were also enriched for C-KIT positive cells and lacked expression of CD34 as identified in other species. The small, agranular SP fraction had high CFU potential after long-term culture with canine bone marrow stromal cells and cytokine supplementation. Yet, canine SP cells demonstrated low-level engraftment within the NOD/SCID-β2m-/- xenotransplantation model as compared to unfractionated canine bone marrow, which was indicative of suboptimal activation of quiescent canine SP cells within the murine bone marrow niche. A second source of transplantable canine stem cells was examined through the derivation of canine embryonic stem cells (cESCs). The cESC lines described herein were determined to have similar pluripotent stem cell characteristics to human embryonic stem cells, in that they were maintained in an undifferentiated state upon extended passaging as determined by their expression of the human stem cell markers, OCT3/4, NANOG, SOX2, SSEA3, SSEA4, TRA1-60, TRA1-81 and alkaline phosphatase. In addition, cESCs could be induced to differentiate to cells of the three germ layers within in vitro embryoid body cultures and adherent differentiation cultures. Importantly, these cESC lines were the first reported to differentiate in vivo within teratomas. One method of transplanting stem cells to canine recipients involves the delivery of donor cells to the yolk sacs of developing fetuses in utero. Utilizing cells labeled with supraparamagnetic particles conjugated to a Dragon Green fluorophore and the intracellular fluorescent dye, CMTMR, donor cells were tracked from the yolk sac injection site to fetal tissues after transplantation in early (day-25) and mid (day-35) gestation canine fetuses. Labeled cells were localized primarily to the fetal liver and developing bone marrow cavities when examined at gestational day 32, and had been redistributed to not only the fetal liver and bone marrow by day 42, but also to nonhematopoietic tissues, including the lungs and hearts. No labeled cells were detected within the yolk sacs of transplanted fetuses at either time point. These studies demonstrated the efficacy of yolk sac in utero transplantation for the delivery of donor cells to fetal tissues. Collectively, these results indicate that canine stem cells with characteristics similar to human can be isolated and their engraftment, proliferation and differentiation may be assessed in future studies utilizing the canine in utero transplantation model employing yolk sac delivery.

Background: Multipotent mesenchymal stromal cells (MSC) can be recovered from a variety of tissues in the body. Yet, their functional properties were shown to vary depending on tissue origin. While MSC have emerged as a favoured cell type for tendon regenerative therapies, very little is known about the influence of the MSC source on their properties relevant to tendon regeneration. The aim of this study was to assess and compare the expression of tendon extracellular matrix proteins and tendon differentiation markers in MSC derived from different sources as well as in native tendon tissue. MSC isolated from equine bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood and tendon tissue were characterized and then subjected to mRNA analysis by real-time polymerase chain reaction. Results: MSC derived from adipose tissue displayed the highest expression of collagen 1A2, collagen 3A1 and decorin compared to MSC from all other sources and native tendon tissue (p < 0.01). Tenascin-C and scleraxis expressions were highest in MSC derived from cord blood compared to MSC derived from other sources, though both tenascin-C and scleraxis were expressed at significantly lower levels in all MSC compared to native tendon tissue (p < 0.01). Conclusions: These findings demonstrate that the MSC source impacts the cell properties relevant to tendon regeneration. Adipose derived MSC might be superior regarding their potential to positively influence tendon matrix reorganization.