Annelise Bennaceur-Griscelli

Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model

Background Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. Results We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. Conclusions These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment.

A Boost of BMP4 Accelerates the Commitment of Human Embryonic Stem Cells to the Endothelial Lineage

Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular‐like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain‐containing receptor (KDR) and CD144/KDR. Because the CD133+KDR+ population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144+KDR+ population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144+KDR+ peak was shifted to day 7. Most of these cells, however, were CD31−, becoming CD31+ in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4‐boosted day 7, CD144+KDR+CD31− population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12. STEM CELLS 2009;27:1750–1759

TLR Ligands Stimulation Protects MSC from NK Killing

Mesenchymal stem cells (MSCs) play a fundamental role in allograft rejection and graft‐versus‐host disease through their immunosuppressive abilities. Recently, Toll‐like receptors (TLR) have been shown to modulate MSC functions. The aim of this study was to investigate the effects of several TLR ligands on the interaction between MSC and natural killer (NK) cells. Our results show that TLR‐primed adult bone marrow and embryonic MSC are more resistant than unprimed MSC to IL‐2‐activated NK‐induced killing. Such protection can be explained by the modulation of Natural Killer group 2D ligands major histocompatibility complex class I chain A and ULBP3 and DNAM‐1 ligands by TLR‐primed MSC. These results indicate that MSCs are able to adapt their immuno‐behavior in an inflammatory context, decreasing their susceptibility to NK killing. In addition, TLR3 but not TLR4‐primed MSC enhance their suppressive functions against NK cells. However, the efficiency of this response is heterogeneous, even if the phenotypes of different analyzed MSC are rather homogeneous. The consequences could be important in MSC‐mediated cell therapy, since the heterogeneity of adult MSC responders may be explored in order to select the more efficient responders. Stem Cells 2014;32:290–300

A Cross‐Talk Between Stromal Cell‐Derived Factor‐1 and Transforming Growth Factor‐β Controls the Quiescence/Cycling Switch of CD34+ Progenitors Through FoxO3 and Mammalian Target of Rapamycin

Cell cycle regulation plays a fundamental role in stem cell biology. A balance between quiescence and proliferation of hematopoietic stem cells in interaction with the microenvironment is critical for sustaining long‐term hematopoiesis and for protection against stress. We analyzed the molecular mechanisms by which stromal cell‐derived factor‐1 (SDF‐1) exhibited a cell cycle‐promoting effect and interacted with transforming growth factor‐β (TGF‐β), which has negative effects on cell cycle orchestration of human hematopoietic CD34+ progenitor cells. We demonstrated that a low concentration of SDF‐1 modulated the expression of key cell cycle regulators such as cyclins, cyclin‐dependent kinase inhibitors, and TGF‐β target genes, confirming its cell cycle‐promoting effect. We showed that a cross‐talk between SDF‐1‐ and TGF‐β‐related signaling pathways involving phosphatidylinositol 3‐kinase (PI3K)/Akt phosphorylation participated in the control of CD34+ cell cycling. We demonstrated a pivotal role of two downstream effectors of the PI3K/Akt pathway, FoxO3a and mammalian target of rapamycin, as connectors in the SDF‐1‐/TGF‐β‐induced control of the cycling/quiescence switch and proposed a model integrating a dialogue between the two molecules in cell cycle progression. Our data shed new light on the signaling pathways involved in SDF‐1 cell cycle‐promoting activity and suggest that the balance between SDF‐1‐ and TGF‐β‐activated pathways is critical for the regulation of hematopoietic progenitor cell cycle status.

Amniotic Fluid-Derived Mesenchymal Stem Cells Prevent Fibrosis and Preserve Renal Function in a Preclinical Porcine Model of Kidney Transplantation

It is well known that ischemia/reperfusion injuries strongly affect the success of human organ transplantation. Development of interstitial fibrosis and tubular atrophy is the main deleterious phenomenon involved. Stem cells are a promising therapeutic tool already validated in various ischemic diseases. Amniotic fluid‐derived mesenchymal stem cells (af‐MSCs), a subpopulation of multipotent cells identified in amniotic fluid, are known to secrete growth factors and anti‐inflammatory cytokines. In addition, these cells are easy to collect, present higher proliferation and self‐renewal rates compared with other adult stem cells (ASCs), and are suitable for banking. Consequently, af‐MSCs represent a promising source of stem cells for regenerative therapies in humans. To determine the efficiency and the safety of af‐MSC infusion in a preclinical porcine model of renal autotransplantation, we injected autologous af‐MSCs in the renal artery 6 days after transplantation. The af‐MSC injection improved glomerular and tubular functions, leading to full renal function recovery and abrogated fibrosis development at 3 months. The strong proof of concept generated by this translational porcine model is a first step toward evaluation of af‐MSC‐based therapies in human kidney transplantation.

Dual SP/ALDH Functionalities Refine the Human Hematopoietic Lin−CD34+CD38− Stem/Progenitor Cell Compartment†‡§

Identification of prevalent specific markers is crucial to stem/progenitor cell purification. Determinants such as the surface antigens CD34 and CD38 are traditionally used to analyze and purify hematopoietic stem/progenitor cells (HSCs/HPCs). However, the variable expression of these membrane antigens poses some limitations to their use in HSC/HPC purification. Techniques based on drug/stain efflux through the ATP‐binding cassette (ABC)G2 pump (side population [SP] phenotype) or on detection of aldehyde dehydrogenase (ALDH) activity have been independently developed and distinguish the SP and ALDHBright (ALDHBr) cell subsets for their phenotype and proliferative capability. In this study, we developed a multiparametric flow cytometric method associating both SP and ALDH activities on human lineage negative (Lin−) bone marrow cells and sorted different cell fractions according to their SP/ALDH activity level. We find that Lin−CD34+CD38Low/− cells are found throughout the spectrum of ALDH expression and are enriched especially in ALDHBr cells when associated with SP functionality (SP/ALDHBr fraction). Furthermore, the SP marker identified G0 cells in all ALDH fractions, allowing us to sort quiescent cells regardless of ALDH activity. Moreover, we show that, within the Lin−CD34+CD38−ALDHBr population, the SP marker identifies cells with higher primitive characteristics, in terms of stemness‐related gene expression and in vitro and in vivo proliferative potential, than the Lin−CD34+ CD38−ALDHBr main population cells. In conclusion, our study shows that the coexpression of SP and ALDH markers refines the Lin−CD34+CD38− hematopoietic compartment and identifies an SP/ALDHBr cell subset enriched in quiescent primitive HSCs/HPCs. STEM CELLS 2009;27:2552–2562

A major role of TGF-beta1 in the homing capacities of murine hematopoietic stem cell/progenitors.

Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with major in vitro effects on hematopoietic stem cells (HSCs) and lymphocyte development. Little is known about hematopoiesis from mice with constitutive TGF-beta1 inactivation largely because of important embryonic lethality and development of a lethal inflammatory disorder in TGF-beta1(-/-) pups, making these studies difficult. Here, we show that no sign of the inflammatory disorder was detectable in 8- to 10-day-old TGF-beta1(-/-) neonates as judged by both the number of T-activated and T-regulator cells in secondary lymphoid organs and the level of inflammatory cytokines in sera. After T-cell depletion, the inflammatory disease was not transplantable in recipient mice. Bone marrow cells from 8- to 10-day-old TGF-beta1(-/-) neonates showed strikingly impaired short- and long-term reconstitutive activity associated with a parallel decreased in vivo homing capacity of lineage negative (Lin(-)) cells. In addition an in vitro-reduced survival of immature progenitors (Lin(-) Kit(+) Sca(+)) was observed. Similar defects were found in liver cells from TGF-beta1(-/-) embryos on day 14 after vaginal plug. These data indicate that TGF-beta1 is a critical regulator for in vivo homeostasis of the HSCs, especially for their homing potential.

Identification of spectral modifications occurring during reprogramming of somatic cells.

Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However, research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly, this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose.

ATAD3B is a human embryonic stem cell specific mitochondrial protein, re-expressed in cancer cells, that functions as dominant negative for the ubiquitous ATAD3A.

Here we report on the identification of a human pluripotent embryonic stem cell (hESC) specific mitochondrial protein that is re-expressed in cancer cells, ATAD3B. ATAD3B belongs to the AAA+ ATPase ATAD3 protein family of mitochondrial proteins specific to multicellular eukaryotes. Using loss- and gain-of-function approaches, we show that ATAD3B associates with the ubiquitous ATAD3A species, negatively regulates the interaction of ATAD3A with matrix nucleoid complexes and contributes to a mitochondria fragmentation phenotype. We conclude that ATAD3B is a negative regulator of ATAD3A and may function as an adaptor of mitochondrial homeostasis and metabolism in hESCs and cancer cells.

The HOXB4 Homeoprotein Promotes the Ex Vivo Enrichment of Functional Human Embryonic Stem Cell-Derived NK Cells

Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEB-derived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNγ and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34+CD45RA+ precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells.