A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells.

Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine.

Reversal of hyperglycemia by insulin-secreting rat bone marrow- and blastocyst-derived hypoblast stem cell-like cells.

ß-cell replacement may efficiently cure type 1 diabetic (T1D) patients whose insulin-secreting ß-cells have been selectively destroyed by autoantigen-reactive T cells. To generate insulin-secreting cells we used two cell sources: rat multipotent adult progenitor cells (rMAPC) and the highly similar rat extra-embryonic endoderm precursor (rXEN-P) cells isolated under rMAPC conditions from blastocysts (rHypoSC). rMAPC/rHypoSC were sequentially committed to definitive endoderm, pancreatic endoderm, and ß-cell like cells. On day 21, 20% of rMAPC/rHypoSC progeny expressed Pdx1 and C-peptide. rMAPCr/HypoSC progeny secreted C-peptide under the stimulus of insulin agonist carbachol, and was inhibited by the L-type voltage-dependent calcium channel blocker nifedipine. When rMAPC or rHypoSC differentiated d21 progeny were grafted under the kidney capsule of streptozotocin-induced diabetic nude mice, hyperglycemia reversed after 4 weeks in 6/10 rMAPC- and 5/10 rHypoSC-transplanted mice. Hyperglycemia recurred within 24 hours of graft removal and the histological analysis of the retrieved grafts revealed presence of Pdx1-, Nkx6.1- and C-peptide-positive cells. The ability of both rMAPC and HypoSC to differentiate to functional ß-cell like cells may serve to gain insight into signals that govern ß-cell differentiation and aid in developing culture systems to commit other (pluripotent) stem cells to clinically useful ß-cells for cell therapy of T1D.

Early steps in neural development.

We studied early neurulation events in vitro by transplanting quail Hensen's node, central prenodal regions (before the nodus as such develops), or upper layer parts of it on the not yet definitively committed upper layer of chicken anti-sickle regions (of unincubated blastoderms), eventually associated with central blastoderm fragments. We could demonstrate by this quail-chicken chimera technique that after the appearance of a pronounced thickening of the chicken upper layer by the early inductive effect of neighboring endophyll, a floor plate forms by insertion of Hensen's node-derived quail cells into the median part of the groove. This favors, at an early stage, the floor plate "allocation" model that postulates a common origin for notochord and median floor plate cells from the vertebrate's secondary major organizer (Hensen's node in this case). A comparison is made with results obtained after transplantation of similar Hensen's nodes in isolated chicken endophyll walls or with previously obtained results after the use of the grafting procedure in the endophyll walls of whole chicken blastoderms.

Development of separated germ layers of rodent embryos on ectopic sites: a reappraisal.

The method of separation of germ layers of rodent embryos by treating the embryonic shields with proteolytic enzymes and by microsurgery with the subsequent transplantation to ectopic sites has helped to gain a more detailed insight into what is going on during gastrulation in mammals. The space under the kidney capsule of adult animals seems to be the most appropriate ectopic site for transplantation of early postimplantation rat embryos or separated germ layers. After transplantation the grafts develop into teratomas whose complex histological structure reflects the initial developmental capacities of the graft. At the pre-primitive streak and the early primitive streak stages the primitive ectoderm differentiates into tissue derivatives of all three definitive germ layers, often in complex organotypic combinations. This is indirect evidence that all cells of the embryonic body originate from the primitive embryonic ectoderm. Halves of the primitive ectoderm obtained by a longitudinal or transverse cut through the egg cylinder give the same result. At the head fold stage the capacity for differentiation of the ectoderm is restricted to ectodermal and mesodermal derivatives. One day before gastrulation the isolated primitive ectoderm is not able to differentiate as renal isograft. The mesoderm isolated at the head fold stage and at later stages when its segmentation occurs, differentiates almost exclusively into the brown adipose tissue. The embryonic endoderm differentiates only in combination with the mesoderm. After transplantation the embryonic ectoderm loses its epithelial organization and breaks up into a mass of mesenchyme-like cells in which epithelial structures subsequently appear and differentiate in a way reminiscent of the reaggregation of cells in mixed cell suspension in vitro.

Dorsalization and neural induction: properties of the organizer in Xenopus laevis.

We have studied the action of the organizer in Xenopus laevis using grafts labelled with horseradish peroxidase (HRP). Orthotropic grafts of the dorsal marginal zone (the organizer) from an HRP-labelled embryo into an unlabelled host showed that this region contributes to the anterior archenteron wall, to the entire craniocaudal extent of the notochord and to a few cells in the somites. Little or no contribution was made to the neural tube. Orthotopic grafts of the ventral marginal zone (the tissue that responds to a grafted organizer) indicated that it only contributes to the posterior half of the embryo. Within this region it spreads around the entire ventrolateral mesoderm, occasionally contributing a few cells to the somites. The posterior endoderm was also heavily labelled. When the dorsal marginal zone from an HRP-labelled embryo was inserted into a slit cut in the ventral marginal zone of an unlabelled host a mirror-symmetrical double-dorsal duplicated embryo resulted, in which only the notochord and a few cells in the somites of the secondary embryo were derived from the graft. The bulk of the secondary somites was, therefore, derived from host ventral marginal zone tissue which normally makes very little contribution to the somites. This indicates that host ventral marginal zone becomes dorsalized by the graft. The neural tube of the secondary embryo was also unlabelled, showing that it was induced by the influence of the graft on the overlying ectoderm, which normally forms ventral epidermis. We have also grafted ventral marginal zone tissue into a slit cut into the dorsal marginal zone of a host embryo. HRP-labelled tissue was grafted into an unlabelled embryo and vice versa. This graft did not produce a double ventral embryo and this reinforces the traditional view that the dorsal marginal zone is a special signalling region. Instead, the resulting embryos usually had a twinned notochord with the graft tissue in between, differentiated as somite. This confirms that juxtaposing ventral and dorsal marginal zone 'dorsalizes' the ventral tissue but does not affect the dorsal tissue which differentiates, as usual, as notochord. Thus, our results allow us to conclude that the organizer mediates two distinct interactions in bringing about the formation of duplicated embryos. The first is dorsalization of adjacent ventral mesoderm and the second is the induction of neuroepithelium from ectoderm overlying the new archenteron roof.

Histogenetic and neoplastic potential of different regions of the mouse embryonic egg cylinder.

The histogenetic and neoplastic potentials of defined regions of the 8th day mouse embryonic egg cylinder were examined following ectopic transfer to beneath the testis capsule. No differences in histogenetic potential were detected between anterior and posterior slices of the embryo, either when composed of all three germ layers or of embryonic ectoderm alone. Small anterior and distal fragments of embryonic ectoderm also produced similar histogenetic profiles, although posterior fragments failed to grow in this ectopic site. The histogenetic potential of anterior and distal fragments exceeded the developmental fate ascribed to these two regions in the embryo (Beddington, 1981). There was some evidence for regionalization with respect to neoplastic potential, anterior slices of the embryo giving rise to a higher incidence of embryonal carcinoma cells than posterior slices.