In multicellular organisms specific stem cell types with distinct developmental potentials occur during development. Transient pluripotent stem cells, which can differentiate into derivatives of all three germ layers (endoderm, ectoderm and mesoderm), are generated during blastocyst development. Adult stem cells, developing at later stages, are more restricted in their potential, since they can differentiate into progenitors and mature effector cell types of only one stem cell system. Adult stem cells have been identified in a variety of tissues in the adult organism and are important for lifelong tissue homeostasis and repair.

Stem cells are functionally defined by two unique attributes: their high self-renewal capacity and their multilineage differentiation potential. The presence of both characteristics in one cell is rare and sets these highly specialized cells aside from the majority of the somatic cell populations. These unique properties make stem cells ideal targets for approaches of gene and cell replacement therapy.

stem_4

Embryonic stem cells (ES cells) give rise to cells of all three germ layers, including hematopoietic stem cells (HSC). HSC develop into all mature blood cells. The laboratory has a particular interest in dendritic cells (DC).

Gene Expression and Epigenetic Signatures of Hematopoetic Stem Cells

Maintenance of all hematopoietic cells in our organism is achieved by the continuous growth and differentiation of a population of multipotent hematopoietic stem cells. We study human and mouse hematopoietic stem/progenitor cells and their differentiation potential. We employ transcriptional profiling with DNA microarrays to determine the gene expression repertoire of stem/progenitor cells and their differentiated progeny (Hacker et al., 2003; Hieronymus et al., 2005; Zenke and Hieronymus, 2006; Felker et al., 2010). Epigenetic signatures of cells are determined by chromatin immunoprecipitation and next generation sequencing (ChIP-Seq) to elucidate how epigenetic signatures determine stemness and differentiation potential (Chauvistré et al., 2014; Lin et al., 2015).

 

Stem Cell Engineering

iPS cells provide unique opportunities for disease modeling, drug development and cell therapy. However, frequently their differentiation potential is rather poor, in particular towards mesodermal lineages, such as hematopoietic cells. We used cell fusion of iPS cells with hematopoietic stem cells to increase the propensity and differentiation potential of pluripotent stem cells towards hematopoietic cells and other mesendodermal lineages, such as cardiomyocytes, hepatocytes and endothelial cells (Qin et al., 2014).

Immunogenicity of iPS cells and iPS cell-derived cells remains controversial. Sertoli cells constitute the structural framework in testis and provide an immune-privileged environment for germ cells. We found that early-passage Ser-iPS cells retain some somatic memory of Sertoli cells that confers reduced immunogenicity of iPS cells and iPS cell-derived cells in vivo and in vitro (Wang et al., 2014). Our data suggest that immune-privileged Sertoli cells represent a preferred source for iPS cell generation if it comes to the use of iPS cell-derived cells for transplantation.

 

CRISPR-Cas9 Human IRF8-/- iPS cells and IRF8-/- ES cells are obtained by CRISPR/Cas9 genome editing and differentiated into hematopoietic progenitors and their progeny (cDC1 and cDC2, classical dendritic cells type 1 and 2, respectively; pDC, plasmacytoid dendritic cells).  

iPS cells and precision genome engineering with CRISPR/Cas are particularly well suited for cell engineering. We used CRISPR/Cas technology to generated human iPS cells deficient in IRF8 (interferon regulatory factor 8). IRF8 is a lineage determining transcription factor in hematopoiesis and IRF8-/- iPS cell-derived hematopoietic cells are deficient in development of specific DC subsets and in DC function (Sontag et al., 2017a, 2017b). IRF8-/- iPS cells now allows studying human immune deficiency in vitro, including the pathophysiology of IRF8 deficient DC.

IRF8 (A) Human IRF8-/- and IRF8+/+ iPS cells (left) are induced to differentiate into hematopoietic progenitors and their differentiated progeny (right). (B) Gene expression in IRF8-/- and IRF8+/+ hematopoietic cells depicted in heat map format (red, high and blue, low gene expression) 

To meet the need of obtaining large numbers of iPS cells for disease modeling and drug development, a consortium of experts in engineering sciences and stem cell biology develops an automatic production system for iPS cells, referred to as StemCellFactory (www.stemcellfactory.net, www.stemcellfactory.org).

In this context we employ CRISPR/Cas technology to generate human iPS cells and ES cells with disease specific mutations for disease modeling and drug screening (see also German Stem Cell Cores).

 

StemCellFactory Poster Thumbnail    

EFRE Logo

StemCellFactory III: Dieses Vorhaben wurde aus Mitteln des Europäischen Fonds für regionale Entwicklung (EFRE) gefördert

     

Selected Publications

Differentiation of human induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) into dendritic cell (DC) subsets.
Sontag, S., Förster, M., Seré, K. and Zenke, M. (2017b).
Bio-protocol 7, e2419.
 
Modelling IRF8 deficient human hematopoiesis and dendritic cell development with engineered induced pluripotent stem cells.
Sontag, S., Förster, M., Qin, J., Wanek, P., Mitzka, S., Schüler, H. M., Koschmieder, S., Rose-John, S., Seré, K. and Zenke, M. (2017a).
Stem Cells 35, 898-908.
 
Reduced immunogenicity of induced pluripotent stem cells derived from Sertoli cells.
Wang, X., Qin, J., Zhao, R.C., and Zenke. M. (2014).
PLoS One 9, e106110.
 
Cell fusion enhances mesendodermal differentiation of human induced pluripotent stem cells.
Qin, J., Sontag, S., Lin, Q., Mitzka, S., Leisten, I., Schneider, R.K., Wang, X., Jauch, A., Peitz, M., Brüstle, O., Wagner, W., Zhao, R.C., and Zenke, M. (2014).
Stem Cells Dev. 23, 2875-2882.
 
The polycomb protein Ezh2 impacts on induced pluripotent stem cell generation.
Ding, X., Wang, X., Sontag, S., Qin, J., Wanek, P., Lin, Q., and Zenke, M. (2014).
Stem Cells Dev. 23, 931-940.
 
Polycomb group protein Bmi1 promotes hematopoietic cell development from ES cells.
Ding, X., Lin, Q., Ensenat-Waser, R., Rose-John, S., and Zenke, M. (2012).
Stem Cells Dev. 21, 121-132.
 
Human adult germline stem cells in question.
Ko, K., Araúzo-Bravo, M. J., Tapia, N., Kim, J., Lin, Q., Bernemann, C., Han, D. W., Gentile, L., Reinhardt, P., Greber, B., Schneider, R. K., Kliesch, S., Zenke, M., and Schöler, H. R. (2010).
Nature 465, E1-E3.
 
Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors.
Kim, J. B., Zaehres, H., Wu, G., Gentile, L., Ko, K., Sebastiano, V., Arauzo-Bravo J. M., Ruau, D., Han, D. W., Zenke, M., and Schöler H. R. (2008).
Nature, 454, 646-650.
 
Pluripotency associated genes are reactivated by chromatin modifying agents in neurosphere cells.
Ruau, D., Ensenat-Waser, R., Dinger, T. C., Vallabhapurapu, D. S., Rolletschek, A., Hacker, C., Hieronymus, T., Wobus, A. M., Müller, A. M., and Zenke, M. (2008).
Stem Cells 26, 920-926.