Multilineage readout after HSC expansion – erythrocytes matter

KEYWORDS:

• erythroid
• human HSC expansion
• megakaryocyte
• MSCs
• multilineage potential

This article refers to:

Human mesenchymal and murine stromal cells support human lympho-myeloid progenitor expansion but not maintenance of multipotent haematopoietic stem and progenitor cells

Haematopoietic stem cell (HSC) transplantation can cure numerous haematopoietic disorders including certain leukemias and anemias. However, the limited number of HLA-matched 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.^1,2 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 HSCs³ or to branch off early during differentiation.⁴ 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 macrophages⁵ or the use of Kit-mutant recipient mouse models allows for a meaningful read-out of human erythroid and megakaryocytic lineage potential (⁶, unpublished data).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.⁴ 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.⁷ 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 CD133⁺ CD34⁺ multipotent haematopoietic stem and progenitor cells (HSCs) over a culture period of 2 weeks.⁷ The co-culture supported the expansion of CD133⁺ CD34⁺ 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^low CD34⁺ progenitors but not in expanded CD133⁺ CD34⁺ 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.¹

It is surprising, that MSC lines fail to support the maintenance and/or expansion of multipotent HSCs because MSC-like 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 more successful in maintaining multilineage potential of expanded HSCs.

Taken together, the study by Radtke et al. strikingly emphasizes the importance for a reliable validation of multilineage potential after ex vivo HSC expansion. The use of novel mouse mutants for this approach still awaits validation, and careful in vitro analysis of erythroid and megakaryocytic potential may in the meantime provide a simple but meaningful assay for the expansion of multipotent HSCs.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.