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In adults, stem cells are responsible for the maintenance of many actively renewing tissues, such as haematopoietic, skin, gut and germinal tissues. These stem cells can self-renew or be committed to becoming progenitors. Stem-cell commitment is thought to be irreversible but in male and female Drosophila melanogaster, it was shown recently that differentiating germ cells can revert to functional stem cells that can restore germinal lineage. Whether progenitors are also able to generate stem cells in mammals remains unknown. Here we show that purified mouse spermatogonial progenitors committed to differentiation can generate functional germinal stem cells that can repopulate germ-cell-depleted testes when transplanted into adult mice. We found that GDNF, a key regulator of the stem-cell niche, and FGF2 are able to reprogram in vitro spermatogonial progenitors for reverse differentiation. This study supports the emerging concept that the stem-cell identity is not restricted in adults to a definite pool of cells that self-renew, but that stemness could be acquired by differentiating progenitors after tissue injury and throughout life.

How transcription factors (TFs) cooperate within large protein complexes to allow rapid modulation of gene expression during development is still largely unknown. Here we show that the key haematopoietic LIM-domain-binding protein-1 (LDB1) TF complex contains several activator and repressor components that together maintain an erythroid-specific gene expression programme primed for rapid activation until differentiation is induced. A combination of proteomics, functional genomics and in vivo studies presented here identifies known and novel co-repressors, most notably the ETO2 and IRF2BP2 proteins, involved in maintaining this primed state. The ETO2–IRF2BP2 axis, interacting with the NCOR1/SMRT co-repressor complex, suppresses the expression of the vast majority of archetypical erythroid genes and pathways until its decommissioning at the onset of terminal erythroid differentiation. Our experiments demonstrate that multimeric regulatory complexes feature a dynamic interplay between activating and repressing components that determines lineage-specific gene expression and cellular differentiation.

IntroductionOne of the aims of regenerative medicine is to repair or restore functional organs by engrafting adult or fetal somatic stem cells into a damaged tissue. These cells, present in most self-renewing tissues, such as skin, intestine, and the hematopoietic system, can be purified, expanded ex vivo, and then used for reconstitution of damaged tissues. 1,2 Many major advances have been achieved in purification and ex vivo amplification of somatic stem cells, 1,3 but few data are available on the prerequisites that will enhance their in vivo biologic activities. New methods to improve the efficiency of bone marrow transplantation and, more generally, reconstitution of damaged tissues by somatic stem cells, depend on tracking of stem cells injected into the animal and thus on the development of imaging strategies that reveal the recruitment, homing, and initial proliferation of these injected somatic stem cells in the context of a living body.The best-characterized mammalian adult somatic stem cells are the hematopoietic stem cells (HSCs) 4,5 whose maintenance and development in the bone marrow are dependent on the HSC niche through niche-regulating pathways. 6 HSCs can be purified close to homogeneity, 7 and a single HSC can produce lifelong complete hematopoietic reconstitution of a lethally irradiated recipient mouse. 4 Many adhesion molecules, signaling pathways, and transcription factors that regulate hematopoietic reconstitution have been characterized, and the roles of these regulatory factors have been shown in vivo using overexpression or genetic inactivation.Yet, the critical early events of recruitment to the bone marrow, homing in the bone marrow microenvironment, and initial proliferation of HSCs after transplantation into lethally irradiated mice are poorly characterized because few methods are available to study these dynamics processes at the cellular level.The early cellular events that precede hematopoietic reconstitution from a small number of HSCs cannot be studied in vitro on hematopoietic cells recovered from recipient animals and presents a demanding challenge for imaging studies as the initial signals that can be detected are very weak. Among imaging techniques, 4 can presently be used to follow the reconstitution of the hematopoietic system at the cellular level. The first technique combines local surgery for placement of a bone window that is used for fluorescent microscopy, but this technique is invasive and limited by the size of the window. 8 The second technique combines high-resolution confocal microscopy and 2-photon video imaging and has greatly improved detection of multiple fluorescent signals in a living animal. This intravital microscopy permits high-resolution imaging of small tissue volume but, in the case of hematopoietic reconstitution, is limited by the thickness of the bone. [9][10][11][12] It cannot be used to study hematopoietic reconstitution in long bones and has been used to image hematopoietic reconstitution in mouse calvarium bone marrow. The third technique ...

Human and murine skin wounding commonly results in fibrotic scarring, but the murine wounding model wound-induced hair neogenesis (WIHN) can frequently result in a regenerative repair response. Here, we show in single-cell RNA sequencing comparisons of semi-regenerative and fibrotic WIHN wounds, increased expression of phagocytic/lysosomal genes in macrophages associated with predominance of fibrotic myofibroblasts in fibrotic wounds. Investigation revealed that macrophages in the late wound drive fibrosis by phagocytizing dermal Wnt inhibitor SFRP4 to establish persistent Wnt activity. In accordance, phagocytosis abrogation resulted in transient Wnt activity and a more regenerative healing. Phagocytosis of SFRP4 was integrin-mediated and dependent on the interaction of SFRP4 with the EDA splice variant of fibronectin. In the human skin condition hidradenitis suppurativa, phagocytosis of SFRP4 by macrophages correlated with fibrotic wound repair. These results reveal that macrophages can modulate a key signaling pathway via phagocytosis to alter the skin wound healing fate.

Ionizing radiation (IR) exposure causes rapid and acute bone marrow (BM) suppression that is reversible for nonlethal doses. Evidence is accumulating that IR can also provoke long-lasting residual hematopoietic injury. To better understand these effects, we analyzed phenotypic and functional changes in the stem/progenitor compartment of irradiated mice over a 10-week period. We found that hematopoietic stem cells (HSCs) identified by their repopulating ability continued to segregate within the Hoechst dye excluding ''side population (SP)'' early after IR exposure. However, transient phenotypic changes were observed within this cell population: Sca-1 (S) and c-Kit (K) expression levels were increased and severely reduced, respectively, with a concurrent increase in the proportion of SP SK cells positive for established indicators of the presence of HSCs: CD150 and CD105. Ten weeks after IR exposure, expression of Sca-1 and c-Kit at the SP cell surface returned to control levels, and BM cellularity of irradiated mice was restored. However, the c-Kit 1 Sca-1 1 Lin 2/low (KSL) stem/progenitor compartment displayed major phenotypic modifications, including an increase and a severe decrease in the frequencies of CD150 1 Flk2 2 and CD150 2 Flk2 1 cells, respectively. CD150 1 KSL cells also showed impaired reconstituting ability, an increased tendency to apoptosis, and accrued DNA damage. Finally, 15 weeks after exposure, irradiated mice, but not agematched controls, allowed engraftment and significant hematopoietic contribution from transplanted congenic HSCs without additional host conditioning. These results provide novel insight in our understanding of immediate and delayed IR-induced hematopoietic injury and highlight similarities between HSCs of young irradiated and old mice.

Crosstalk between transcription factors and cytokines precisely regulates tissue homeostasis. Transcriptional intermediary factor 1γ (TIF1γ) regulates vertebrate hematopoietic development, can control transcription elongation, and is a component of the TGF-β signaling pathway. Here we show that deletion of TIF1γ in adult hematopoiesis is compatible with life and long-term maintenance of essential blood cell lineages. However, loss of TIF1γ results in deficient long-term hematopoietic stem cell (LT-HSC) transplantation activity, deficient short-term HSC (ST-HSC) bone marrow retention, and priming ST-HSCs to myelomonocytic lineage. These defects are hematopoietic cell-autonomous, and priming of TIF1γ-deficient ST-HSCs can be partially rescued by wild-type hematopoietic cells. TIF1γ can form complexes with TAL1 or PU.1-two essential DNA-binding proteins in hematopoiesis-occupy specific subsets of their DNA binding sites in vivo, and repress their transcriptional activity. These results suggest a regulation of adult hematopoiesis through TIF1γ-mediated transcriptional repression of TAL1 and PU.1 target genes.

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