Epigenetic changes to human umbilical cord blood cells cultured with three proteins indicate partial reprogramming to a pluripotent state.

We have previously reported the existence of a subpopulation of cells from human umbilical cord blood capable of differentiating into oligodendrocytes, Schwann cells, bone, muscle, and endothelial cells despite their origins as CD45-positive cells. These stem cells (called FSFl cells) arise only after a period in vitro in medium containing FGF4, SCF, and Flt-3 ligand (FSFl medium) during which they express the pluripotency genes Oct4 and Nanog. The objective of this study was to determine if the novel expression of these pluripotency genes coupled with the newly acquired ability of these cells to differentiate into all three germ layers was the result of epigenetic changes to these cells after reprogramming in FSFl medium. We confirm that CD45-derived FSFl cells express Oct4 protein at levels similar to that observed among undifferentiated embryonic stem cells, during which time acetylated histones H3 and H4 display increased binding at the promoter region of Oct4. Changes to binding of acetylated histones at Oct4 when these cells are in a differentiated state (either prior to FSFl culture or after in vitro differentiation into neural cells) and when they are undifferentiated suggest that this is one way by which these cells acquire their pluripotency. While DNA hypermethylation at this gene region as well as the continued H3 and H4 acetylation at the CD45 promoter region among FSFl cells indicate this is only a partial reprogramming event, this is a significant step toward non-transgene reprogramming of somatic cells.

Neural progenitors, neurons and oligodendrocytes from human umbilical cord blood cells in a serum-free, feeder-free cell culture.

We have previously demonstrated that lineage negative cells (Lin(neg)) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone, muscle, endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin(neg) UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin, neurofilament, A2B5 and Sox2) are expressed in Lin(neg) cells cultured in FGF4, SCF, Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase, GalC, Olig2 and the late-stage marker MOSP are observed, as is the Schwann cell marker PMP22. In summary, Lin(neg) UCB cells, when appropriately cultured, are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation.

Bone marrow transplantation results in human donor blood cells acquiring and displaying mouse recipient class I MHC and CD45 antigens on their surface.

BACKGROUND: Mouse models of human disease are invaluable for determining the differentiation ability and functional capacity of stem cells. The best example is bone marrow transplants for studies of hematopoietic stem cells. For organ studies, the interpretation of the data can be difficult as transdifferentiation, cell fusion or surface antigen transfer (trogocytosis) can be misinterpreted as differentiation. These events have not been investigated in hematopoietic stem cell transplant models. METHODOLOGY/PRINCIPAL FINDINGS: In this study we investigated fusion and trogocytosis involving blood cells during bone marrow transplantation using a xenograft model. We report that using a standard SCID repopulating assay almost 100% of the human donor cells appear as hybrid blood cells containing both mouse and human surface antigens. CONCLUSION/SIGNIFICANCE: Hybrid cells are not the result of cell-cell fusion events but appear to be due to efficient surface antigen transfer, a process referred to as trogocytosis. Antigen transfer appears to be non-random and includes all donor cells regardless of sub-type. We also demonstrate that irradiation preconditioning enhances the frequency of hybrid cells and that trogocytosis is evident in non-blood cells in chimera mice.

Identification and analysis of in vitro cultured CD45-positive cells capable of multi-lineage differentiation.

We report on a subset of cells that co-purify with CD45-positive/Lineage minus (CD45(pos)/Lin(minus)) hematopoietic cells that are capable of in vitro differentiation into multi-potential cells including cells with neuroectoderm properties. Although these cells are CD45 positive and have properties similar to CD45-negative mesenchymal progenitor cells (MPC) derived from bone marrow (BM), they are neither hematopoietic cells nor mesenchymal cells. These CD45(pos)/Lin(minus) cells can be expanded in vitro, express the stem cell genes Oct-4 and Nanog and can be induced to differentiate into endothelial cells, osteoblasts, muscle cells and neural cells at frequencies similar to those reported for bone marrow mesenchymal cells. Long-term culture of these cells followed by transplantation into NOD/SCID mice resulted in positive bone marrow stromal cell engraftment but not hematopoietic engraftment, suggesting that despite their CD45-positive status these cells do not have the same properties as hematopoietic stem cells. Clonal cell analysis determined that the culture period caused a broadening in the differentiation potential of the starting population.

Limb developmental stages of the newt Notophthalmus viridescens.

The current study consists of a detailed description of normal larval forelimb and hindlimb development in the newt, Notophthalmus viridescens. This is the first comprehensive record of staging limb development in this urodele amphibian species, augmenting Fankhauser's work which was published in fragments 60 years ago. Larvae were obtained by natural spawning, without hormone injections and while experimental conditions such as temperature and light cycles were kept constant (20 degrees C and 12L/12D), larval sizes have been added here merely as guidelines as development is influenced by a number of extrinsic factors. Care has been taken to indicate similarities to either the Harrison or Glucksohn stages, although this relates mainly to their morphological descriptions of the limbs. Observations from limb use are also mentioned in the text where pertinent to the description of development.

Growth and apoptosis during larval forelimb development and adult forelimb regeneration in the newt ( Notophthalmus viridescens).

Many of the genes involved in the initial development of the limb in higher vertebrates are also expressed during regeneration of the limb in urodeles such as Notophthalmus viridescens. These similarities have led researchers to conclude that the regeneration process is a recapitulation of development, and that patterning of the regenerate mimics pattern formation in development. However, the developing limb and the regenerating limb do not look similar. In developing urodele forelimbs, digits appear sequentially as outgrowths from the limb palette. In regeneration, all the digits appear at once. In this work, we address the issue of whether regeneration and development are similar by examining growth and apoptosis patterns. In contrast to higher vertebrates, forelimb development in the newt, N. viridescens, does not use interdigital apoptosis as the method of digit separation. During adult forelimb regeneration, apoptosis seems to play an important role in wound healing and again during cartilage to bone turnover in the advanced digits and radius/ulna. However, similar to forelimb development, demarcation of the digits in adult forelimb regeneration does not involve interdigital apoptosis. Outgrowth, rather than regression of the interdigital mesenchyme, leads to the individualization of forelimb digits in both newt development and regeneration.