Dormant and self-renewing hematopoietic stem cells and their niches

In the mouse, over the last 20 years, a set of cell-surface markers and activities have been identified, enabling the isolation of bone marrow (BM) populations highly enriched in hematopoietic stem cells (HSCs). These HSCs have the ability to generate multiple lineages and are capable of long-term self-renewal activity such that they are able to reconstitute and maintain a functional hematopoietic system after transplantation into lethally irradiated recipients. Using single-cell reconstitution assays, various marker combinations can be used to achieve a functional HSC purity of almost 50%. Here we have used the differential expression of six of these markers (Sca1, c-Kit, CD135, CD48, CD150, and CD34) on lineage-depleted BM to refine cell hierarchies within the HSC population. At the top of the hierarchy, we propose a dormant HSC population (Lin(-)Sca1(+)c-Kit(+) CD48(-)CD150(+)CD34(-)) that gives rise to an active self-renewing CD34(+) HSC population. HSC dormancy, as well as the balance between self-renewal and differentiation activity, is at least, in part, controlled by the stem cell niches individual HSCs are attached to. Here we review the current knowledge about HSC niches and propose that dormant HSCs are located in niches at the endosteum, whereas activated HSCs are in close contact to sinusoids of the BM microvasculature.

Long-Term Engraftment of Primary Bone Marrow Stromal Cells Repairs Niche Damage and Improves Hematopoietic Stem Cell Transplantation

Zuzana Tatárová

Hematopoietic stem cell (HSC) transplantation represents a curative treatment for various hematological disorders. However, delayed reconstitution of innate and adaptive immunity often causes fatal complications. HSC maintenance and lineage differentiation are supported by stromal niches, and we now find that bone marrow stroma cells (BMSCs) are severely and permanently damaged by the pre-conditioning irradiation required for efficient HSC transplantation. Using mouse models, we show that stromal insufficiency limits the number of donor-derived HSCs and B lymphopoiesis. Intra-bone transplantation of primary, but not cultured, BMSCs quantitatively reconstitutes stroma function in vivo, which is mediated by a multipotent NT5E(+) (CD73)(+) ENG(-) (CD105)(-) LY6A(+) (SCA1)(+) BMSC subpopulation. BMSC co-transplantation doubles the number of functional, donor-derived HSCs and significantly reduces clinically relevant side effects associated with HSC transplantation including neutropenia and humoral immunodeficiency. These data demonstrate the potential of stroma recovery to improve HSC transplantation.

Cell Press2017

The role of recruited bone-marrow derived mesenchymal stem cells in the establishment of the tumor stroma and a growth-supportive environment

Jean-Paul Abbühl

The Mesenchymal Stem Cell (MSC) is a self-renewing multipotent progenitor originally found in the bone marrow. It has drawn strong interest from translational research because of the multipotency of MSCs, their immunosuppression, their intrinsic homing and their promotion of wound healing in human patients. Currently available methods rely on in vitro culture for the isolation and expansion of MSCs and most studies used these cells for their investigation. Fibroblasts constitute the connective tissue and support epithelial or hematopoietic cells. Although they have already been extensively investigated, it is not clear whether stem cells exist in vivo which specifically regenerate fibroblasts. MSCs are a potential candidate, because of their fibroblastic shape in vitro, their differentiation towards several mesodermal lineages and their supportive function. We aim to characterize the in vivo differentiation potential of MSCs toward fibroblasts in normal and tumor tissues. A new transplantation approach was developed in order to transfer labeled MSCs in the bone marrow and assess their recruitment and lineage contribution in vivo. Long-term engraftment was accomplished by isolating and transplanting these cells within one day. To this end, strategies for their purification, preconditioning of the host and transplantation methodologies were established during this thesis. We were able to enrich MSCs over 1000 fold by combining a new extraction protocol, a negative selection by MACS and a positive selection by FACS. Cloning of sorted MSCs was performed to confirm their multipotency at single cell level. GFP-expressing MSCs were able to engraft in host mice tolerant for GFP and their expansion in vivo was stimulated by block of GSK3 activity. Altogether, the chimeric MSC model was used to track the recruitment of bone-marrow derived MSCs into spontaneous breast tumors. The tumor microenvironment encompasses several non-tumorigenic cells interacting with cancer cells. Among them, cancer-associated fibroblasts are known to participate in carcinogenesis. Whilst this heterogeneous stromal population has been extensively investigated, their source(s) remain elusive. I now propose tumoral MSCs as one source of cancer-associated fibroblast, based on their ability to self-renew and reconstitute stromal heterogeneity upon serial transplantation. Finally, I introduced the new concept that the multipotency of MSCs is subjugated by cancer cells to meet their requirements for enhanced tumor progression and prepare for metastasis. I found MSCs present in the tumor microenvironment to differentiate into cancer-associated osteoblasts and produce hydroxyapatite-mineralization in vivo. Microcalcification is an accepted poor prognostic feature in breast cancer patients but its function and origin is not documented. I showed that hydroxyapatite was able to promote proliferation of tumor cells and increased intracellular calcium concentration. Lastly, cancer-associated osteoblast and hydroxyapatite were able to promote tumor growth and metastasis in vivo. To conclude, bone marrow derived MSCs are circulating stem cells that participate in the establishment of the pathogenic stroma in breast cancer. Owing to their multipotency, MSCs give rise to cancer-associated osteoblasts that induce pro-malignant mineralization in situ. Subsequently, cancer cells harbor increased intracellular calcium concentration, enhanced proliferation and metastasis. We foresee the development of new stroma-targeted therapies that prevent the mineralization of tumor tissue and reduce metastasis in breast cancer patients.

EPFL2014

The Cell Differentiation Status Influences the Outcome of Notch-Induced Malignancy

Caroline Poisson

The Notch signaling cascade is a well conserved pathway that regulates many aspects of embryonic development and plays a crucial role in the differentiation process and tissue homeostasis in multiple organs of adult species. Several human pathological disorders, including cancer, arise as a consequence of the deregulation of the Notch signaling pathway. Notch deregulation, up to date, has been best characterized in acute T-cell lymphoblastic leukemia (T-ALL). Indeed, in T-ALL pediatric patients activating Notch1 mutations have been identified in more than 50% of the cases. Increased knowledge of the mechanisms underlying T-ALL development and maintenance is clearly needed as one out of 5 patients with T-ALL has a very poor prognosis. Moreover, the high toxicity of chemotherapeutic regimens has been associated with elevated risks of developing severe secondary health problems. In the present study, we characterized the role of Notch in T-ALL induction and maintenance. Using transgenic mouse models, we could demonstrate that the oncogenic potential of Notch is dependent on RBP-Jκ transcriptional activity. Indeed, the canonical Notch signaling pathway, mediated by the RBP-Jκ transcription factor, is essential for both Notch-mediated T-ALL induction and maintenance, as well as for the induction of a Notch-mediated lymphoproliferative disorder. Moreover, constitutive overexpression of Notch1 at discrete developmental stages within the hematopoietic system, led us to identify a correlation between the differentiation status of a cell, within the hierarchy of the hematopoietic tree, and its sensitivity to Notch1-induced transformation. We demonstrated that forced Notch1 expression in hematopoietic stem cells was not sufficient to induce T-ALL. Nevertheless, aberrant Notch1 expression in the bone marrow resulted in the development of a lethal lymphoproliferative disorder. Overexpression of Notch1 in thmus at the DN2 to DN3 stage of T-cell delvelopment generated an aggressive transplantable T-ALL with important metastatic potential. This suggested that progenitors already committed to the T-cell lineage are highly susceptible to Notch-mediated transformation, which is relevant to the human disease. Nevertheless, forced Notch1 expression after the β-selection checkpoint in the thymys, was no longer sufficient to induce transformation of late DN3 to DN4 thymocytes. We therefore established a murine genetic system that allowed us to dissect the mechanisms of aberrant Notch1 signaling in a lymphoproliferative disorder and a true T-ALL model. We were able to assess the genetic signature that is involved in the transplantability and metastatic potential of leukemic cells and generated a list of candidate genes implicated in these processes. In addition, we correlated genes identified in our murine models with genes associated as well in human T-ALL profiles to identify genes implicated in T-ALL pathogenesis in both mouse and human.