Characterization of gene regulatory networks underlying key properties in human hematopoietic stem cell ontogeny.

Human hematopoiesis starts at early yolk sac and undergoes site- and stage-specific changes over development. The intrinsic mechanism underlying property changes in hematopoiesis ontogeny remains poorly understood. Here, we analyzed single-cell transcriptome of human primary hematopoietic stem/progenitor cells (HSPCs) at different developmental stages, including yolk-sac (YS), AGM, fetal liver (FL), umbilical cord blood (UCB) and adult peripheral blood (PB) mobilized HSPCs. These stage-specific HSPCs display differential intrinsic properties, such as metabolism, self-renewal, differentiating potentialities etc. We then generated highly co-related gene regulatory network (GRNs) modules underlying the differential HSC key properties. Particularly, we identified GRNs and key regulators controlling lymphoid potentiality, self-renewal as well as aerobic respiration in human HSCs. Introducing selected regulators promotes key HSC functions in HSPCs derived from human pluripotent stem cells. Therefore, GRNs underlying key intrinsic properties of human HSCs provide a valuable guide to generate fully functional HSCs in vitro.

Revisiting the lineage contribution of hematopoietic stem and progenitor cells.

For a long time, self-renewing and multipotent hematopoietic stem cells (HSCs) have been thought to make a major contribution to both embryonic and adult hematopoiesis. The canonical hematopoietic hierarchy illustrating HSC self-renewal and multipotency has been established mainly based on invasive functional assays (e.g. transplantation or colony-forming units in the spleen and in culture), which evaluate the cellular potentials of HSCs. With the extensive applications of non-invasive cell fate-mapping strategies, recent lineage tracing-based studies have suggested that not all native hematopoiesis is established via the hierarchical differentiation of HSCs. By contrast, hematopoietic progenitor cells (HPCs) are a dominant contributor to both embryonic and young adult hematopoiesis. These new findings help redefine the cellular origins of embryonic and adult hematopoiesis under native conditions, and emphasize the differences in revealing HSC potential versus HSC fate using distinct approaches during stress and native hematopoiesis. Here, we review recent advances in HPC and HSC development, and provide an updated perspective to incorporate these new findings with our traditional understanding of developmental and adult hematopoiesis.

Chemically-Defined, Xeno-Free, Scalable Production of hPSC-Derived Definitive Endoderm Aggregates with Multi-Lineage Differentiation Potential.

For the production and bio-banking of differentiated derivatives from human pluripotent stem cells (hPSCs) in large quantities for drug screening and cellular therapies, well-defined and robust procedures for differentiation and cryopreservation are required. Definitive endoderm (DE) gives rise to respiratory and digestive epithelium, as well as thyroid, thymus, liver, and pancreas. Here, we present a scalable, universal process for the generation of DE from human-induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs). Optimal control during the differentiation process was attained in chemically-defined and xeno-free suspension culture, and high flexibility of the workflow was achieved by the introduction of an efficient cryopreservation step at the end of DE differentiation. DE aggregates were capable of differentiating into hepatic-like, pancreatic, intestinal, and lung progenitor cells. Scale-up of the differentiation process using stirred-tank bioreactors enabled production of large quantities of DE aggregates. This process provides a useful advance for versatile applications of DE lineages, in particular for cell therapies and drug screening.

Recent insights into the naïve state of human pluripotency and its applications.

The past decade has seen significant interest in the isolation of pluripotent stem cells corresponding to various stages of mammalian embryonic development. Two distinct and well-defined pluripotent states can be derived from mouse embryos: "naïve" pluripotent cells with properties of pre-implantation epiblast, and "primed" pluripotent cells, resembling post-implantation epiblast. Prompted by the successful interconversion between these two stem cell states in the mouse system, several groups have devised strategies for inducing a naïve state of pluripotency in human pluripotent stem cells. Here, we review recent insights into the naïve state of human pluripotency, focusing on two methods that confer defining transcriptomic and epigenomic signatures of the pre-implantation embryo. The isolation of naïve human pluripotent stem cells offers a window into early developmental mechanisms that cannot be adequately modeled in primed cells, such as X chromosome reactivation, metabolic reprogramming, and the regulation of hominid-specific transposable elements. We outline key unresolved questions regarding naïve human pluripotency, including its extrinsic and intrinsic control mechanisms, potential for embryonic and extraembryonic differentiation, and general utility as a model system for human development and disease.

Clonal and Quantitative In Vivo Assessment of Hematopoietic Stem Cell Differentiation Reveals Strong Erythroid Potential of Multipotent Cells.

Hematopoiesis is arguably one of the best understood stem cell systems; however, significant challenges remain to reach a consensus understanding of the lineage potential, heterogeneity, and relationships of hematopoietic stem and progenitor cell populations. To gain new insights, we performed quantitative analyses of mature cell production from hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor populations. Assessment of the absolute numbers of mature cell types produced by each progenitor cell revealed a striking erythroid dominance of all myeloid-competent progenitors assessed, accompanied by strong platelet reconstitution. All populations with myeloid potential also produced robust numbers of red blood cells and platelets in vivo. Clonal analysis by single-cell transplantation and by spleen colony assays revealed that a significant fraction of HSCs and multipotent progenitors have multilineage potential at the single-cell level. These new insights prompt an erythroid-focused model of hematopoietic differentiation.

Loss of CD44dim Expression from Early Progenitor Cells Marks T-Cell Lineage Commitment in the Human Thymus.

Human T-cell development is less well studied than its murine counterpart due to the lack of genetic tools and the difficulty of obtaining cells and tissues. Here, we report the transcriptional landscape of 11 immature, consecutive human T-cell developmental stages. The changes in gene expression of cultured stem cells on OP9-DL1 match those of ex vivo isolated murine and human thymocytes. These analyses led us to define evolutionary conserved gene signatures that represent pre- and post-αß T-cell commitment stages. We found that loss of dim expression of CD44 marks human T-cell commitment in early CD7+CD5+CD45dim cells, before the acquisition of CD1a surface expression. The CD44-CD1a- post-committed thymocytes have initiated in frame T-cell receptor rearrangements that are accompanied by loss of capacity to differentiate toward myeloid, B- and NK-lineages, unlike uncommitted CD44dimCD1a- thymocytes. Therefore, loss of CD44 represents a previously unrecognized human thymocyte stage that defines the earliest committed T-cell population in the thymus.

A Transient Developmental Hematopoietic Stem Cell Gives Rise to Innate-like B and T Cells.

The generation of distinct hematopoietic cell types, including tissue-resident immune cells, distinguishes fetal from adult hematopoiesis. However, the mechanisms underlying differential cell production to generate a layered immune system during hematopoietic development are unclear. Using an irreversible lineage-tracing model, we identify a definitive hematopoietic stem cell (HSC) that supports long-term multilineage reconstitution upon transplantation into adult recipients but does not persist into adulthood in situ. These HSCs are fully multipotent, yet they display both higher lymphoid cell production and greater capacity to generate innate-like B and T lymphocytes as compared to coexisting fetal HSCs and adult HSCs. Thus, these developmentally restricted HSCs (drHSCs) define the origin and generation of early lymphoid cells that play essential roles in establishing self-recognition and tolerance, with important implications for understanding autoimmune disease, allergy, and rejection of transplanted organs.

NG2-glia and their functions in the central nervous system.

In the central nervous system, NG2-glia represent a neural cell population that is distinct from neurons, astrocytes, and oligodendrocytes. While in the past the main role ascribed to these cells was that of progenitors for oligodendrocytes, in the last years it has become more obvious that they have further functions in the brain. Here, we will discuss some of the most current and highly debated issues regarding NG2-glia: Do these cells represent a heterogeneous population? Can they give rise to different progenies, and does this change under pathological conditions? How do they respond to injury or pathology? What is the role of neurotransmitter signaling between neurons and NG2-glia? We will first give an overview on the developmental origin of NG2-glia, and then discuss whether their distinct properties in different brain regions are the result of environmental influences, or due to intrinsic differences. We will then review and discuss their in vitro differentiation potential and in vivo lineage under physiological and pathological conditions, together with their electrophysiological properties in distinct brain regions and at different developmental stages. Finally, we will focus on their potential to be used as therapeutic targets in demyelinating and neurodegenerative diseases. Therefore, this review article will highlight the importance of NG2-glia not only in the healthy, but also in the diseased brain.

Immortalization and characterization of mouse temporomandibular joint disc cell clones with capacity for multi-lineage differentiation.

OBJECTIVE: Despite the importance of temporomandibular joint (TMJ) disc in normal function and disease, studying the responses of its cells has been complicated by the lack of adequate characterization of the cell subtypes. The purpose of our investigation was to immortalize, clone, characterize and determine the multi-lineage potential of mouse TMJ disc cells. DESIGN: Cells from 12-week-old female mice were cultured and immortalized by stable transfection with human telomerase reverse transcriptase (hTERT). The immortalized cell clones were phenotyped for fibroblast- or chondrocyte-like characteristics and ability to undergo adipocytic, osteoblastic and chondrocytic differentiation. RESULTS: Of 36 isolated clones, four demonstrated successful immortalization and maintenance of stable protein expression for up to 50 passages. Two clones each were initially characterized as fibroblast-like and chondrocyte-like on the basis of cell morphology and growth rate. Further the chondrocyte-like clones had higher mRNA expression levels of cartilage oligomeric matrix protein (COMP) (>3.5-fold), collagen X (>11-fold), collagen II expression (2-fold) and collagen II:I ratio than the fibroblast-like clones. In contrast, the fibroblast-like clones had higher mRNA expression level of vimentin (>1.5-fold), and fibroblastic specific protein 1 (>2.5-fold) than the chondrocyte-like clones. Both cell types retained multi-lineage potential as demonstrated by their capacity to undergo robust adipogenic, osteogenic and chondrogenic differentiation. CONCLUSIONS: These studies are the first to immortalize TMJ disc cells and characterize chondrocyte-like and fibroblast-like clones with retained multi-differentiation potential that would be a valuable resource in studies to dissect the behavior of specific cell types in health and disease and for tissue engineering.

After repeated division, bone marrow stromal cells express inhibitory factors with osteogenic capabilities, and EphA5 is a primary candidate.

The differentiation capability of human bone marrow stromal cells (hBMSCs) is thought to deteriorate over multiple doubling processes. To clarify the deterioration mechanisms, the multilineage differentiation capabilities of short- and long-term passaged BMSCs were compared. Predictably, long-term passaged BMSCs showed reduced differentiation capacities compared to short-term passaged cells. Furthermore, a non-human primate heterotopic bone formation model demonstrated that long-term passaged BMSCs have bone formation capabilities but also exert inhibitory effects on bone formation. This finding indicated that long-term passaged BMSCs express higher levels of inhibitory factors than short-term passaged BMSCs do. Co-culture assays of short- and long-term passaged BMSCs suggested that the inhibitory signals required cell-cell contact and would therefore be expressed on the cell membrane. A microarray analysis of BMSCs identified ephrin type-A receptor 5 (EphA5) as an inhibitory factor candidate. Quantitative PCR revealed that among all members of the ephrin and Eph receptor families, only the expression of EphA5 was increased by BMSC proliferation. A gene knockdown analysis using siRNAs demonstrated that knockdown of EphA5 gene expression in long-term passaged BMSCs led to an increase in ALP mRNA expression. These results indicate that EphA5 may be a negative regulator of bone formation. A better understanding of the roles of the ephrin and Eph receptor families in hBMSCs may lead to alternative approaches for manipulating hBMSC fate. In addition, this avenue of discovery may provide new therapeutic targets and quality-control markers of the osteogenic differentiation capabilities of hBMSCs.