Maria Isabel D. Rossi

Identification of very early lymphoid precursors in bone marrow and their regulation by estrogen

Estrogen is a negative regulator of lymphopoiesis and provides an experimental tool for probing relationships between lymphocyte precursors and stem cells. We found that expression of lymphocyte-associated genes and immunoglobulin (Ig) gene rearrangement occurred before CD45R acquisition. Lymphoid-restricted progenitors that were Lin−IL-7Rα+c-kitloTdT+ (lineage marker−, interleukin receptor 7α+, c-kitlo and terminal deoxynucleotidyl transferase+) were selectively depleted in estrogen-treated mice; within a less differentiated Lin−c-kithi fraction, functional precursors of B and T, but not myeloid, cells were also selectively depleted. TdT and an Ig heavy chain transgene were detected within a hormone-regulated Lin−c-kithiSca-1+CD27+Flk-2+IL-7Rα− subset of this multipotential progenitor population. Identification of these extremely early lymphoid precursors should facilitate investigation of the molecular mechanisms that control lineage-fate decisions in hematopoiesis.

Early B‐lymphocyte precursors and their regulation by sex steroids

Summary: This review describes an improved characterization of early B‐lymphocyte precursors in mice and the remarkable sensitivity of the same cells to hormones. The nuclear enzyme terminal deoxynucleotidyl transferase (TdT) was used as a marker to image and characterize bone marrow cells lacking all lineage‐associated markers. Most early TdT+ precursors have a distinctive density of c‐kit and express the interleukin‐7Ra chain, as well as flt‐3/flk2, but lack CD34. An understanding of those cell surface properties made it possible to obtain highly enriched, viable cells with the potential to give rise to CD19+ lymphocytes in culture. A series of other flow cytometry and culture experiments suggested a possible differentiation sequence for these early pro‐B cells. This new model was used to advantage in our studies of sex steroids. It appears that early precursors represent a hormone‐sensitive control point for determining numbers of new B lymphocytes that are produced within bone marrow. We also compare and contrast these findings with B lymphopoiesis in humans.

Nature or nurture? Steady-state lymphocyte formation in adults does not recapitulate ontogeny

Summary:  Substantial progress has been made in determining developmental relationships between lymphocyte precursors and those corresponding to other blood cell lineages. Indeed, exploitation of RAG1/GFP knock‐in mice has recently made it possible to chart the entire sequence of lymphocyte differentiation events in adult bone marrow and thymus. However, the differentiation pathways proposed for fetal life are very different from this model. We review many examples where the results of gene targeting experiments are substantially dependent on developmental age. In mice, adult patterns of gene expression and corresponding properties of lymphocyte precursors are not fully established until several weeks after birth, and the same might be true for humans. Furthermore, examples are cited where fetal hematopoietic cells did not efficiently acquire those properties when transplanted to an adult environment. There are several important implications of these findings. Cognizance of developmental age‐related changes might resolve apparent conflicts in the literature. Hematopoietic stem cells and their lymphoid lineage progeny appear in waves, and a direct connection is yet to be established between fetal stem cells and ones that sustain adult blood cell formation. There is the possibility that adult stem cells derive from founders with an unknown origin.

Relatively Normal Human Lymphopoiesis but Rapid Turnover of Newly Formed B Cells in Transplanted Nonobese Diabetic/SCID Mice

Human B lineage lymphocyte precursors in chimeric nonobese diabetic/SCID mice transplanted with umbilical cord blood cells were directly compared with those present in normal bone marrow. All precursor subsets were represented and in nearly normal proportions. Cell cycle activity and population dynamics were investigated by staining for the Ki-67 nuclear Ag as well as by incorporation experiments using 5-bromo-2′-deoxyuridine. Again, this revealed that human B lymphopoiesis in chimeras parallels that in normal marrow with respect to replication and progression through the lineage. Moreover, sequencing of Ig gene rearrangement products showed that a diverse repertoire of VH genes was utilized by the newly formed lymphocytes but there was no evidence for somatic hypermutation. The newly formed B cells frequently acquired the CD5 Ag and had a short life span in the periphery. Thus, all molecular requirements for normal B lymphocyte formation are present in nonobese diabetic/SCID mice, but additional factors are needed for recruitment of B cells into a fully mature, long-lived pool. The model can now be exploited to learn about species restricted and conserved environmental cues for human B lymphocyte production.

Lymphoid lineage cells in adult murine bone marrow diverge from those of other blood cells at an early, hormone-sensitive stage.

Advances in cell sorting and GFP knock-in technology have made it possible to identify rare hematopoietic cells in murine bone marrow that are undergoing lymphocyte fate specification. Steroid hormones also represent important research tools for investigating relationships between different categories of lympho-hematopoietic precursors. By selectively blocking entry into and progression within lymphoid lineages, the hormones probably have a major influence on numbers of lymphocytes that are produced under normal circumstances. These issues are discussed within the context of developmental age-dependent changes that occur in the lymphopoietic process.

PS1/γ-Secretase-Mediated Cadherin Cleavage Induces β-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

Re-evaluation of B lymphocyte lineage differentiation schemes

Differentiation models for hematopoietic stem cells are to experimental hematologists what metabolic pathway charts are to biochemists. It has been extremely useful to consider the results of gene targeting and other experiments within the context of diagrams predicting key steps in B and T lymphopoiesis. Comparable schemes for human cells allow assignment of malignant cells to particular differentiation stages and interpretation of immunodeficiencies. Practical advances in multi-parameter flow cytometry and commercially available monoclonal antibodies further account for the popularity of differentiation schemes depicted on the walls of most laboratories. However, there is reason to believe that these models are not accurate or sufficiently complete in all respects. We will briefly recount how our recent studies forced re-evaluation of some widely accepted milestones for murine B lineage differentiation.