Stefanie Liedtke

A New Human Somatic Stem Cell from Placental Cord Blood with Intrinsic Pluripotent Differentiation Potential

Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 1015 cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals.

Oct4 expression revisited: potential pitfalls for data misinterpretation in stem cell research.

Abstract The octamer-binding transcription factor 4 gene encodes a nuclear protein (Oct4, also known as Pou5F1 and Oct3/4) that belongs to a family of transcription factors containing the POU DNA-binding domain. Expression can be detected in embryonic stem cells as well as in adult stem cells, such as bone marrow-derived mesenchymal stem cells. Expression of Oct4 is downregulated coincident with stem cell differentiation and loss of expression leading to differentiation. A role for maintaining pluripotency and self-renewal of embryonic stem cells is ascribed to Oct4 as a pluripotency marker. Results describing Oct4 expression in differentiated cells, including peripheral blood mononuclear cells (PBMCs), neonatal and adult stem cells, as well as cancer cells, must be interpreted with caution. In several publications, Oct4 has been ascribed a function in maintaining self-renewal of adult stem cells. In contrast, other publications reported Oct4 expression in human tumor cells. Here, we summarize the recent findings on Oct4 expression and present possibilities and reasons why several false positive results on Oct4 expression still occur in the recent literature. Also, simple solutions are provided to avoid these positive signals.

No Identical "Mesenchymal Stem Cells" at Different Times and Sites: Human Committed Progenitors of Distinct Origin and Differentiation Potential Are Incorporated as Adventitial Cells in Microvessels

Summary A widely shared view reads that mesenchymal stem/stromal cells (“MSCs”) are ubiquitous in human connective tissues, can be defined by a common in vitro phenotype, share a skeletogenic potential as assessed by in vitro differentiation assays, and coincide with ubiquitous pericytes. Using stringent in vivo differentiation assays and transcriptome analysis, we show that human cell populations from different anatomical sources, regarded as “MSCs” based on these criteria and assumptions, actually differ widely in their transcriptomic signature and in vivo differentiation potential. In contrast, they share the capacity to guide the assembly of functional microvessels in vivo, regardless of their anatomical source, or in situ identity as perivascular or circulating cells. This analysis reveals that muscle pericytes, which are not spontaneously osteochondrogenic as previously claimed, may indeed coincide with an ectopic perivascular subset of committed myogenic cells similar to satellite cells. Cord blood-derived stromal cells, on the other hand, display the unique capacity to form cartilage in vivo spontaneously, in addition to an assayable osteogenic capacity. These data suggest the need to revise current misconceptions on the origin and function of so-called “MSCs,” with important applicative implications. The data also support the view that rather than a uniform class of “MSCs,” different mesoderm derivatives include distinct classes of tissue-specific committed progenitors, possibly of different developmental origin.

Induction of pluripotency in human cord blood unrestricted somatic stem cells

OBJECTIVE Generation of induced pluripotent stem (iPS) cells from human cord blood (CB)-derived unrestricted somatic stem cells and evaluation of their molecular signature and differentiation potential in comparison to human embryonic stem cells. MATERIALS AND METHODS Unrestricted somatic stem cells isolated from human CB were reprogrammed to iPS cells using retroviral expression of the transcription factors OCT4, SOX2, KLF4, and C-MYC. The reprogrammed cells were analyzed morphologically, by quantitative reverse transcription polymerase chain reaction, genome-wide microRNA and methylation profiling, and gene expression microarrays, as well as in their pluripotency potential by in vivo teratoma formation in severe combined immunodeficient mice and in vitro differentiation. RESULTS CB iPS cells are very similar to human embryonic stem cells morphologically, at their molecular signature, and in their differentiation potential. CONCLUSIONS Human CB-derived unrestricted somatic stem cells offer an attractive source of cells for generation of iPS cells. Our findings open novel perspectives to generate human leukocyte antigen-matched pluripotent stem cell banks based on existing CB banks. Besides the obvious relevance of a second-generation CB iPS cell bank for pharmacological and toxicological testing, its application for autologous or allogenic regenerative cell transplantation appears feasible.

The uptake of Texas Red-BSA in the excretory system of schistosomes and its colocalisation with ER60 promoter-induced GFP in transiently transformed adult males.

The excretory system of schistosomes has focused some attention during the last years since accumulating evidence suggests that it plays an important role in the host-parasite interaction. Signalling molecules such as phosphatases, but also proteases have been localised in the excretory system. To some extent, however, localisation studies are limited by the fact that sections of fixed specimens are used. In this study, we tested the fluorescent molecules FITC-dextran and Texas Red-BSA for their ability to enter the excretory system of living Schistosoma mansoni males. It is demonstrated that the dyes selectively stain the excretory tubules which are widely distributed along the worm body. This finding was used to investigate whether the staining of worms with Texas Red-BSA can help to localise transgene activity in worms which were transiently transformed by particle bombardment. A vector was used for transformation which contained the green fluorescent protein gene, under the control of the regulatory elements of the cysteine protease ER60 gene. After transformation and staining, confocal laser scanning microscopy revealed that ER60-induced green fluorescent protein activity colocalises with Texas Red-BSA in the excretory tubules. The results suggest a role for ER60 during the host-parasite interaction. Furthermore, the colocalisation approach introduced here opens further perspectives to characterise gene-expression profiles in this parasite.

Cord Blood for Tissue Regeneration

Umbilical cord blood (CB) has become a commonly accepted source of hematopoietic stem cells for transplantation in children and adults. It is readily available and outperforms bone marrow (BM) as well as peripheral blood stem cells in terms of tolerance for HLA‐mismatches between donor and recipient and its decreased graft‐versus‐host disease. Clinical use has been expanded from hematological malignancies to various areas such as treatment of metabolic genetic disorders or to induce angiogenesis. For the last years CB has been under intense experimental investigation in in vitro differentiation models as well as in preclinical animal models. Since CB‐derived stem cells offer multiple advantages over adult stem cells from other sources like BM, CB may provide a future source of stem cells for tissue repair and regeneration. To facilitate the use of CB‐derived stem cells in clinical scenarios, the biology of these cells needs to be further explored in detail particularly with regard to the fact that different non‐hematopoietic stem cell populations occur within CB. Here we explore the most consistent and the most contradictory data referring to the differentiation potential of CB‐derived stem cells and give an outlook on their potential clinical value including and possible reprogramming into IPS cells. J. Cell. Biochem. 108: 762–768, 2009.

Comparing the Gene Expression Profile of Stromal Cells from Human Cord Blood and Bone Marrow: Lack of the Typical “Bone” Signature in Cord Blood Cells

With regard to the bone-regenerative capacity, bone marrow stromal cells (BMSC) can still be termed the “gold standard.” Nevertheless, neonatal stromal cells from cord blood (CB) feature advantages concerning availability, immaturity, and proliferation potential. The detailed gene expression analysis and overexpression of genes expressed differentially provide insight into the inherent capacity of stromal cells. Microarray and qRT-PCR analyses revealed closely related gene expression patterns of two stromal cell populations derived from CB. In contrast to the CB-derived cell types, BMSC displayed high expression levels of BSP, OSX, BMP4, OC, and PITX2. Lentiviral overexpression of BSP but not of OSX in CB-cells increased the capacity to form a mineralized matrix. BMP4 induced the secretion of proteoglycans during chondrogenic pellet culture and extended the osteogenic but reduced the adipogenic differentiation potential. BMSC revealed the typical osteogenic gene expression signature. In contrast, the CB-derived cell types exhibited a more immature gene expression profile and no predisposition towards skeletal development. The absence of BSP and BMP4—which were defined as potential key players affecting the differentiation potential—in neonatal stromal cells should be taken into consideration when choosing a cell source for tissue regeneration approaches.

SLP65 deficiency results in perpetual V(D)J recombinase activity in pre-B-lymphoblastic leukemia and B-cell lymphoma cells.

Perpetual V(D)J recombinase activity involving multiple DNA double-strand break events in B-cell lineage leukemia and lymphoma cells may introduce secondary genetic aberrations leading towards malignant progression. Here, we investigated defective negative feedback signaling through the (pre-) B-cell receptor as a possible reason for deregulated V(D)J recombinase activity in B-cell malignancy. On studying 28 cases of pre-B-lymphoblastic leukemia and 27 B-cell lymphomas, expression of the (pre-) B-cell receptor-related linker molecule SLP65 (SH2 domain-containing lymphocyte protein of 65 kDa) was found to be defective in seven and five cases, respectively. SLP65 deficiency correlates with RAG1/2 expression and unremitting VH gene rearrangement activity. Reconstitution of SLP65 expression in SLP65-deficient leukemia and lymphoma cells results in downregulation of RAG1/2 expression and prevents both de novo VH–DJH rearrangements and secondary VH replacement. We conclude that iterative VH gene rearrangement represents a frequent feature in B-lymphoid malignancy, which can be attributed to SLP65 deficiency in many cases.

Neonatal mesenchymal-like cells adapt to surrounding cells.

Hematopoietic cord blood (CB) transplantations are performed to treat patients with life-threatening diseases. Besides endothelial cells, the neonatal multipotent stromal cell subpopulations CDSCs (CB-derived stromal cells) and USSCs (unrestricted somatic stromal cells) are like bone marrow (BM) SCs interesting candidates for clinical applications if detailed knowledge is available. Clonal USSC compared to CDSC and BMSC lines differ in their developmental origin reflected by a distinct HOX expression. About 20 (out of 39) HOX genes are expressed in CDSCs (HOX+), whereas native USSCs reveal no HOX gene expression (HOX-). Moreover, USSCs display a lineage-specific absence of the adipogenic differentiation potential. As the specific HOX code can be ascribed to topographic bodysites it may be important to match the HOX code of transplanted cells to the tissue of interest. Herein co-culture experiments were performed, presenting a novel approach to modulate the differentiation potency of USSCs towards HOX positive stromal cells. After co-culturing native USSCs with CDSCs and BMSCs, USSCs adapt a positive HOX code and gain the adipogenic differentiation capacity. These results present for the first time modulation of a lineage-specific differentiation potential by co-culture. Finally, USSCs can be claimed as potential candidates to substitute unique progenitor cell populations in clinical approaches.

Oxygen tension modifies the ‘stemness’ of human cord blood-derived stem cells

BACKGROUND AIMS Amongst different stem cell populations derived from human cord blood (CB), unrestricted somatic stem cells (USSC) are distinguished from CB mesenchymal stromal cells (CB MSC) by expression patterns of homeobox (HOX) genes, delta-like1 homolog (DLK1) expression and adipogenic differentiation potential. In this study we investigated the effects of oxygen tension on the generation, proliferation and expression of stem cell marker genes, which could be critical during large-scale cell culture for clinical applications. METHODS We cultured CB-derived stem cells at 5% and 20% O(2). Telomere length shortening was analyzed and we investigated gene expression using reverse-transcription (RT)-polymerase chain reaction (PCR) and real-time PCR. Additionally we performed adipogenic and osteogenic in vitro differentiation. Results. Altering the cultivation conditions of USSC or CB MSC from 20% to 5% O(2) had no significant impact. In contrast, cell populations derived from primary cultures prepared at 5% O(2) qualified as neither USSC nor as CB MSC. When converted to 20%, their proliferation was diminished, telomere shortening was accelerated, and two of six cell lines ceased expression of HOX genes. The HOX code of the other cell populations was not been affected by culture conditions. CONCLUSIONS Altering culture conditions during generation can impact cell characteristics such as the HOX code. These effects need to be considered when dealing with cell cultures for clinical applications.