Nicole Mende

Kit Regulates HSC Engraftment across the Human-Mouse Species Barrier

In-depth analysis of the cellular and molecular mechanisms regulating human HSC function will require a surrogate host that supports robust maintenance of transplanted human HSCs in vivo, but the currently available options are problematic. Previously we showed that mutations in the Kit receptor enhance engraftment of transplanted HSCs in the mouse. To generate an improved model for human HSC transplantation and analysis, we developed immune-deficient mouse strains containing Kit mutations. We found that mutation of the Kit receptor enables robust, uniform, sustained, and serially transplantable engraftment of human HSCs in adult mice without a requirement for irradiation conditioning. Using this model, we also showed that differential KIT expression identifies two functionally distinct subpopulations of human HSCs. Thus, we have found that the capacity of this Kit mutation to open up stem cell niches across species barriers has significant potential and broad applicability in human HSC research.

Clonal expansion capacity defines two consecutive developmental stages of long-term hematopoietic stem cells.

Hematopoietic stem cells expressing intermediate levels of Kit have superior repopulation capacity after transplantation compared with those expressing high levels of Kit.

CCND1–CDK4–mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo

Maintenance of stem cell properties is associated with reduced proliferation but it is unknown whether the transition kinetics through distinct cell cycle phases influences the function of HSCs. Mende et al examine the effects of increasing two cell cycle complexes CCND1–CDK4 and CCNE1–CDK2 on the transition kinetics of human HSCs and their maintenance and functional alterations in vivo.

Niche WNT5A regulates the actin cytoskeleton during regeneration of hematopoietic stem cells.

Here, we show that the Wnt5a-haploinsufficient niche regenerates dysfunctional HSCs, which do not successfully engraft in secondary recipients. RNA sequencing of the regenerated donor Lin− SCA-1+ KIT+ (LSK) cells shows dysregulated expression of ZEB1-associated genes involved in the small GTPase-dependent actin polymerization pathway. Misexpression of DOCK2, WAVE2, and activation of CDC42 results in apolar F-actin localization, leading to defects in adhesion, migration and homing of HSCs regenerated in a Wnt5a-haploinsufficient microenvironment. Moreover, these cells show increased differentiation in vitro, with rapid loss of HSC-enriched LSK cells. Our study further shows that the Wnt5a-haploinsufficient environment similarly affects BCR-ABLp185 leukemia-initiating cells, which fail to generate leukemia in 42% of the studied recipients, or to transfer leukemia to secondary hosts. Thus, we show that WNT5A in the bone marrow niche is required to regenerate HSCs and leukemic cells with functional ability to rearrange the actin cytoskeleton and engraft successfully.

Multilineage readout after HSC expansion – erythrocytes matter

Haematopoietic stem cell (HSC) transplantation can cure numerous haematopoietic disorders including certain leukemias and anemias. However, the limited number of HLAmatched donor cells is still a major hurdle for the success of clinical HSC transplantation and extensive attempts have been made to define conditions supporting the ex vivo maintenance or expansion of functional human HSCs, including the use of cytokine-containing cocktails or stromal co-culture systems, stimulating ‘self-renewal’ pathways and maintaining the pre-existing HSC potential. Despite these efforts HSC expansion or even maintenance during culture remains a prominent challenge. The classical concept of hematopoiesis is based on the loss of multilineage potential during sequential binary decisions generating lineage-restricted, and subsequently, unipotent progenitors. The prevailing consensus predicted the presence of oligopotent precursors mainly harboring the propensity to give rise to myeloid, erythroid and megakaryocytic or lymphoid lineages, termed common myeloid progenitors or common lymphoid progenitors, respectively. This strict lineage-specification of early haematopoietic progenitors has recently been challenged, and during human blood cell formation erythroid and megakaryocytic lineage potential is now suggested to be restricted to HSCs or to branch off early during differentiation. These novel perceptions of lineage-commitment need to be considered when searching for ex vivo expansion conditions for human HSCs and, consequently, it should be assayed for myeloid, lymphoid, megakaryocytic, and erythroid lineage potential within the expanded population. Transplantation into immune-deficient mice is currently considered the best surrogate assay to measure sustainable HSC function including engraftment, expansion and multilineage differentiation. However, only the depletion of endogenous murine macrophages or the use of Kit-mutant recipient mouse models allows for a meaningful read-out of human erythroid and megakaryocytic lineage potential (, unpublished data). Consequently, only long-term lympho-myeloid engraftment can be assayed comfortably in mouse models. However, lympho-myeloid engraftment cannot discriminate between the engraftment of true multipotent HSCs and lymphoid-primed multipotent progenitors (LMPPs) that lack erythroid and megakaryocytic lineage potential. These results suggest that in vitro assays may currently be preferred over in vivo studies to also prove megakaryocyte-erythroid potential of expanded HSCs. Based on this idea, Radtke et al. reported recently in Cell Cycle on the use of an in vitro culture system to assay for the maintenance of lineage potential during ex vivo expansion of human HSCs. The authors tested a number of murine and human mesenchymal stromal cells (MSCs) for their capacity to support the maintenance of multilineage differentiation potential of CD133C CD34C multipotent haematopoietic stem and progenitor cells (HSCs) over a culture period of 2 weeks. The co-culture supported the expansion of CD133C CD34C cells, which maintained myeloid and lymphoid differentiation potential as revealed by in vitro colony formation. However, after culture, erythroid colony-forming potential was detectable only in expanded CD133 CD34C progenitors but not in expanded CD133C CD34C multipotent HSPCs, suggesting that this population, after culture, consists of LMPP-like progenitors and not of multipotent HSCs. This observation is consistent with the results of a clinical study where long-term engraftment ( >1 year) was produced primarily by non-expanded but not by simultaneously transplanted expanded cord blood cells, suggesting a depletion of long-term reconstituting multipotent HSCs in MSC co-culture. It is surprising, that MSC lines fail to support the maintenance and/or expansion of multipotent HSCs because MSClike populations expressing nestin, the leptin receptor, or abundant amounts of the chemokine CXCL12, are of major importance for at least murine HSC maintenance in vivo. However, MSCs reside in close vicinity to endothelial cells and the authors suggest that a co-culture approach of HSCs with MSCs in combination with endothelium or other niche cells may be