Emma Rörby

The Tetraspanin CD9 Affords High-Purity Capture of All Murine Hematopoietic Stem Cells

Prospective isolation is critical for understanding the cellular and molecular aspects of stem cell heterogeneity. Here, we identify the cell surface antigen CD9 as a positive marker that provides a simple alternative for hematopoietic stem cell isolation at high purity. Crucially, CD9 affords the capture of all hematopoietic stem cells in murine bone marrow in the absence of contaminating populations that lack authentic stem cell function. Using CD9 as a tool to subdivide hematopoietic stem-cell-containing populations, we provide evidence for heterogeneity at the cellular, functional, and molecular levels.

Human hematopoietic stem/progenitor cells overexpressing Smad4 exhibit impaired reconstitution potential in vivo.

Hematopoietic stem cells (HSCs) constitute a rare population of tissue-specific cells that can self-renew and differentiate into all lineages of the blood cell system. These properties are critical for tissue regeneration and clinical applications of HSCs. Cord blood is an easily accessible source of HSCs. However, the number of HSCs from one unit is too low to effectively transplant most adult patients, and expansion of HSCs in vitro has met with limited success because of incomplete knowledge regarding mechanisms regulating self-renewal. Members of the TGF-β superfamily have been shown to regulate HSCs through the Smad signaling pathway; however, its role in human HSCs has remained relatively uncharted in vivo. Therefore, we asked whether enforced expression of the common-Smad, Smad4, could reveal a role for TGF-β in human hematopoietic stem/progenitor cells (HSPCs) from cord blood. Using a lentiviral overexpression approach, we demonstrate that Smad4 overexpression sensitizes HSPCs to TGF-β, resulting in growth arrest and apoptosis in vitro. This phenotype translates in vivo into reduced HSPC reconstitution capacity yet intact lineage distribution. This suggests that the Smad pathway regulates self-renewal independently of differentiation. These findings demonstrate that the Smad signaling circuitry negatively regulates the regeneration capacity of human HSPCs in vivo.

Hematopoietic stem cells are regulated by Cripto, as an intermediary of HIF‐1α in the hypoxic bone marrow niche

Cripto has been known as an embryonic stem (ES)‐ or tumor‐related soluble/cell membrane protein. In this study, we demonstrated that Cripto has a role as an important regulatory factor for hematopoietic stem cells (HSCs). Recombinant Cripto sustained the reconstitution ability of HSCs in vitro. Flow cytometry analysis uncovered that GRP78, one of the candidate receptors for Cripto, was expressed on a subset of HSCs and could distinguish dormant/myeloid‐biased HSCs and active/lymphoid‐biased HSCs. Cripto is expressed in hypoxic endosteal niche cells where GRP78+ HSCs mainly reside. Proteomics analysis revealed that Cripto‐GRP78 binding stimulates glycolytic metabolism‐related proteins and results in lower mitochondrial potential in HSCs. Furthermore, conditional knockout mice for HIF‐1α, a master regulator of hypoxic responses, showed reduced Cripto expression and decreased GRP78+ HSCs in the endosteal niche area. Thus, Cripto‐GRP78 is a novel HSC regulatory signal mainly working in the hypoxic niche.

SPARC is dispensable for murine hematopoiesis, despite its suspected pathophysiological role in 5q-myelodysplastic syndrome.

SPARC is dispensable for murine hematopoiesis, despite its suspected pathophysiological role in 5q-myelodysplastic syndrome

A network including TGFβ/Smad4, Gata2, and p57 regulates proliferation of mouse hematopoietic progenitor cells

Transforming growth factor β (TGFβ) is a potent inhibitor of hematopoietic stem and progenitor cell proliferation. However, the precise mechanism for this effect is unknown. Here, we have identified the transcription factor Gata2, previously described as an important regulator of hematopoietic stem cell function, as an early and direct target gene for TGFβ-induced Smad signaling in hematopoietic progenitor cells. We also report that Gata2 is involved in mediating a significant part of the TGFβ response in primitive hematopoietic cells. Interestingly, the cell cycle regulator and TGFβ signaling effector molecule p57 was found to be upregulated as a secondary response to TGFβ. We observed Gata2 binding upstream of the p57 genomic locus, and importantly, loss of Gata2 abolished TGFβ-stimulated induction of p57 as well as the resulting growth arrest of hematopoietic progenitors. Our results connect key molecules involved in hematopoietic stem cell self-renewal and reveal a functionally relevant network, regulating proliferation of primitive hematopoietic cells.

The stem cell regulator PEDF is dispensable for maintenance and function of hematopoietic stem cells

Pigment epithelium derived factor (PEDF), a ubiquitously expressed 50 kDa secreted glycoprotein, was recently discovered to regulate self-renewal of neural stem cells and have a supportive effect on human embryonic stem cell growth. Here, we analyzed expression of PEDF in the murine hematopoietic stem cell (HSC) compartments and found that PEDF is highly expressed in primary long-term HSCs. Therefore, we characterized the hematopoietic system in a knockout mouse model for PEDF and using this model we surprisingly found that PEDF is dispensable for HSC regulation. PEDF knockout mice exhibit normal hematopoiesis in steady state conditions and the absence of PEDF lead to normal regeneration capacity in a serial competitive transplantation setting. Additionally, PEDF-deficient cells exhibit unaltered lineage distribution upon serial transplantations. When human cord blood stem and progenitor cells were cultured in media supplemented with recombinant PEDF they did not show changes in growth potential. Taken together, we report that PEDF is not a critical regulatory factor for HSC function during regeneration in vivo or growth of human stem/progenitor cells in vitro.