Albrecht M. Müller

Pluripotency Associated Genes Are Reactivated by Chromatin‐Modifying Agents in Neurosphere Cells

Chromatin architecture in stem cells determines the pattern of gene expression and thereby cell identity and fate. The chromatin‐modifying agents trichostatin A (TSA) and 5‐Aza‐2′‐deoxycytidine (AzaC) affect histone acetylation and DNA methylation, respectively, and thereby influence chromatin structure and gene expression. In our previous work, we demonstrated that TSA/AzaC treatment of neurosphere cells induces hematopoietic activity in vivo that is long‐term, multilineage, and transplantable. Here, we have analyzed the TSA/AzaC‐induced changes in gene expression by global gene expression profiling. TSA/AzaC caused both up‐ and downregulation of genes, without increasing the total number of expressed genes. Chromosome analysis showed no hot spot of TSA/AzaC impact on a particular chromosome or chromosomal region. Hierarchical cluster analysis revealed common gene expression patterns among neurosphere cells treated with TSA/AzaC, embryonic stem (ES) cells, and hematopoietic stem cells. Furthermore, our analysis identified several stem cell genes and pluripotency‐associated genes that are induced by TSA/AzaC in neurosphere cells, including Cd34, Cd133, Oct4, Nanog, Klf4, Bex1, and the Dppa family members Dppa2, 3, 4, and 5. Sox2 and c‐Myc are constitutively expressed in neurosphere cells. We propose a model in which TSA/AzaC, by removal of epigenetic inhibition, induces the reactivation of several stem cell and pluripotency‐associated genes, and their coordinate expression enlarges the differentiation potential of somatic precursor cells.

Stem Cell Plasticity in Mammals and Transdetermination in Drosophila: Common Themes?

Stem cells have been identified in a number of mammalian tissues (e.g., bone marrow, muscle, gut, skin, and neural tissues). Until recently, it was generally believed that the differentiation potential of a mammalian somatic stem cell is restricted to one tissue only, as in the case of hematopoietic stem cells differentiating into hematopoietic cells. In this sense, somatic stem cells are limited in their differentiation potential. Several lines of evidence now challenge the idea of unilateral development. New reports show mammalian somatic stem cells can, in the course of regeneration, repopulate heterologous cell systems and therefore possess a surprisingly broad spectrum of differentiation potential. Thus, mammalian stem cells are apparently capable of fate changes between stem cell systems, although the mechanisms leading to such changes are unclear.

Synergistic effects of growth factors and mesenchymal stromal cells for expansion of hematopoietic stem and progenitor cells

OBJECTIVE The number of hematopoietic stem and progenitor cells (HPCs) per cord blood unit is limited, and this can result in delayed engraftment or graft failure. In vitro expansion of HPCs provides a perspective to overcome these limitations. Cytokines as well as mesenchymal stromal cells (MSCs) have been shown to support HPCs ex vivo expansion, but a systematic analysis of their interplay remains elusive. MATERIALS AND METHODS Twenty different combinations of growth factors (stem cell factor [SCF], thrombopoietin [TPO], fibroblast growth factor-1 [FGF-1], angiopoietin-like 5, and insulin-like growth factor-binding protein 2), either with or without MSC coculture were systematically compared for their ability to support HPC expansion. CD34(+) cells were stained with carboxyfluorescein diacetate N-succinimidyl ester to monitor cell division history in conjunction with immunophenotype. Colony-forming unit frequencies and hematopoietic reconstitution of nonobese diabetic severe combined immunodeficient mice were also assessed. RESULTS Proliferation of HPCs was stimulated by coculture with MSCs. This was further enhanced in combination with SCF, TPO, and FGF-1. Moreover, these conditions maintained expression of primitive surface markers for more than four cell divisions. Colony-forming unit-initiating cells were not expanded without stromal support, whereas an eightfold increase was reached by simultaneous cytokine-treatment and MSC coculture. Importantly, in comparison to expansion without stromal support, coculture with MSCs significantly enhanced hematopoietic chimerism in a murine transplantation model. CONCLUSIONS The supportive effect of MSCs on hematopoiesis can be significantly increased by addition of specific recombinant growth factors; especially in combination with SCF, TPO, and FGF-1.

Replicative aging and differentiation potential of human adipose tissue-derived mesenchymal stromal cells expanded in pooled human or fetal bovine serum

BACKGROUND AIMS Mesenchymal stromal cells (MSC) are promising candidates for innovative cell therapeutic applications. For clinical-scale manufacturing, different supplements have been evaluated as alternatives for the commonly used fetal bovine serum (FBS). We have reported previously that pooled human AB serum (HS) accelerates the proliferation of adipose tissue-derived MSC (ASC) while maintaining key functions of MSC biology such as differentiation, immune suppression and growth factor secretion. ASC expanded in FBS-supplemented culture media undergo replicative aging that is associated with a progressive loss of differentiation capacity but without indications of cellular transformation. The effects of HS media on ASC long-term culture, however, remain poorly characterized. METHODS Long-term cultures of ASC in FBS and HS media were analyzed with respect to proliferation, marker expression, differentiation and immune suppression. RESULTS Despite signs of an accelerated proliferation, extended life span and clonogenic capacity of ASC cultivated in HS-supplemented media, HS and FBS cultures revealed no significant differences with respect to differentiation potential and expression of senescence markers. Anchorage-independent growth, which is indicative of tumorigenic properties, was not observed in either culture conditions. Similarly, immune suppressive activities were maintained. Donor variation regarding differentiation potential and marker expression became apparent in this study independent of the culture supplement or culture duration. CONCLUSIONS We have demonstrated that the use of pooled allogeneic HS maintains the characteristics of ASC even after long-term expansion, further demonstrating that the use of HS is an alternative to FBS.

In vivo haematopoietic activity is induced in neurosphere cells by chromatin‐modifying agents

Modifications of DNA and chromatin are fundamental for the establishment and maintenance of cell type‐specific gene expression patterns that constitute cellular identities. To test whether the developmental potential of fetal brain‐derived cells that form floating sphere colonies (neurospheres) can be modified by destabilizing their epigenotype, neurosphere cells were treated with chemical compounds that alter the acetylation and methylation patterns of chromatin and DNA. Intravenous infusion of bulk or clonally derived neurosphere cells treated with a combination of trichostatin A (TSA) plus 5‐aza‐2′‐deoxycytidine (AzaC) (TSA/AzaC neurosphere cells) yielded long‐term, multilineage and transplantable neurosphere‐derived haematopoietic repopulation. Untreated neurosphere cells exhibited no haematopoietic repopulation activity. The neurosphere‐derived haematopoietic cells showed a diploid karyotype, indicating that they are unlikely to be products of cell fusion events, a conclusion strengthened by multicolour fluorescence in situ hybridization. Our results indicate that altering the epigenotype of neurosphere cells followed by transplantation enables the generation of neurosphere‐derived haematopoietic cells.

Progressive and Controlled Development of Mouse Dendritic Cells from Flt3+CD11b+ Progenitors In Vitro

Dendritic cells (DC) represent key regulators of the immune system, yet their development from hemopoietic precursors is poorly defined. In this study, we describe an in vitro system for amplification of a Flt3+CD11b+ progenitor from mouse bone marrow with specific cytokines. Such progenitor cells develop into both CD11b+ and CD11b− DC, and CD8α+ and CD8α− DC in vivo. Furthermore, with GM-CSF, these progenitors synchronously differentiated into fully functional DC in vitro. This two-step culture system yields homogeneous populations of Flt3+CD11b+ progenitor cells in high numbers and allows monitoring the consecutive steps of DC development in vitro under well-defined conditions. We used phenotypic and functional markers and transcriptional profiling by DNA microarrays to study the Flt3+CD11b+ progenitor and differentiated DC. We report here on an extensive analysis of the surface Ag expression of Flt3+CD11b+ progenitor cells and relate that to surface Ag expression of hemopoietic stem cells. Flt3+CD11b+ progenitors studied exhibit a broad overlap of surface Ags with stem cells and express several stem cell Ags such as Flt3, IL-6R, c-kit/SCF receptor, and CD93/AA4.1, CD133/AC133, and CD49f/integrin α6. Thus, Flt3+CD11b+ progenitors express several stem cell surface Ags and develop into both CD11b+ and CD11b− DC, and CD8α+ and CD8α− DC in vivo, and thus into both of the main conventional DC subtypes.

Mesenchymal stromal cells (MSCs): science and f(r)iction

Due to their multi-lineage differentiation capacity, support of haematopoiesis, immunomodulation and secretion of proregenerative factors, mesenchymal stem/stromal cells (MSCs) are in the focus of intense research since decades. The literature is replete with reports on their potential in preclinical model systems. However, the heterogeneity of the primary cell population as starting material and the diverse protocols for isolation and cultivation are hampering progress in their clinical application. Consensus on common standards and harmonised isolation and characterisation protocols are important to ensure safety and efficacy. This review focuses on the recent scientific evidence of clinically relevant properties and on the speculative cardiomyogenic and hepatic differentiation potential of MSCs. Special emphasis is put on the importance of standardisation and harmonisation in clinical-scale manufacturing.

Genetic instability and diminished differentiation capacity in long-term cultured mouse neurosphere cells

The potential use of neural stem cells in basic research, drug testing and for development of therapeutic strategies requires large scale in vitro amplification, increasing the probability of genetic instability and transformation. Little is known, however, about potential correlations between long-term culture of neural stem and progenitor cells (NSPCs), changed differentiation and self-renewal capacities, and the occurrence of chromosomal instability. This study investigates the effect of extended culture time on self-renewal, differentiation capacity, cell cycle phase distribution, telomere length, telomerase activity and chromosomal stability on fetal brain-derived cells that form floating sphere colonies (neurospheres). We observed that increased sphere-forming capacity indicative of increased proliferation was accompanied by a decreased ability to differentiate into neural lineages. The high mobility group A (Hmga2) gene positively regulates self-renewal via repression of p16(Ink4a) and p19(ARF) gene expression. This study discerned an upregulation of Hmga2 gene and protein expression and decreased p16(Ink4a) and p19(ARF) gene expression, suggesting that Hmga2 might promote the proliferation of neurosphere cells in long-term culture. Further, our analyses revealed a significant decrease in telomere length after 4 weeks of culturing that is paralleled by a moderate upregulation of telomerase activity. Importantly, regular gain of chromosome 1 with random structural chromosomal aberrations was observed within 16 weeks of neurosphere cell culture. Genetic instability and diminished differentiation capacity seem to be a consequence of long-term culture of neurosphere cells. These data indicate the necessity to analyze self-renewal, differentiation capacity, telomere length, tumor suppressor genes and chromosomal stability in neurosphere cultures prior to their usage in basic research, drug testing or the development of therapeutic strategies.

Chimaerism and erythroid marker expression after microinjection of human acute myeloid leukaemia cells into murine blastocysts

It has been suggested that the embryonic microenvironment can control the survival and the transformed phenotype of tumour cells. Here, we addressed the hypothesis that the murine embryonic microenvironment can induce the differentiation of human tumour cells. To examine such interactions, we injected human leukaemic cells into preimplantation murine blastocysts at embryonic day 3.5 of gestation (E3.5). Microinjection of human KG-1 myeloid leukaemia cells and primary human acute myeloid leukaemia (AML) cells led to the generation of chimaeric embryos and adults. We observed that in E12.5 murine embryos, KG-1 cells were preferentially detected in yolk sac and peripheral blood, while primary AML cells mainly seeded the aorta gonad mesonephros region of chimaeric embryos. Analysis of the donor contribution in 15 different adult tissues showed that progeny of primary AML cells seeded to various haematopoietic and nonhaematopoietic tissues. Chimaeric embryos and adults showed no apparent tumour formation. Furthermore, analysis of chimaeric E12.5 embryos revealed that the progeny of human KG-1 cells activated erythroid-specific human globin and glycophorin A expression. In summary, our data indicate that human AML cells activate markers of erythroid differentiation after injection into early murine embryos.

Plasticity of hematopoietic stem cells and cellular memory

Summary: Stem cell systems represent an effective and powerful approach for tissue development and regeneration of diverse tissue types. Common and defining features of these exceptional cells are the capacity for self‐renewal and the potential for differentiation into multiple mature cell types. Recently, surprising new observations have indicated that stem cells isolated from one adult tissue can also give rise to mature cells of other cell lineages, irrespective of classical germ layer designations. This discovery has resulted in quantum leaps in both scientific knowledge and the potential applications of stem cells. The new findings contradict central dogmas of commitment and differentiation of stem and progenitor cells. However, the true potential of somatic stem cells is just emerging and the new findings have to be defined more fully and integrated into a unifying model of stem cell potential and behavior. Here we analyze the developmental potential of hematopoietic stem cells of mouse and man following their injection into the murine preimplantation blastocyst, an environment that allows the development of all cell lineages. In addition, we discuss the emerging lines of evidence of the developmental plasticity of hematopoietic and other somatic stem cells and consider how cellular memory of transcriptional states is established and may be potentially involved in this phenomenon.