Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. These cells are usually culture expanded prior to their application. However, a precise molecular definition of MSC and the sequel of long-term in vitro culture are yet unknown. In this study, we have addressed the impact of replicative senescence on human MSC preparations. Within 43 to 77 days of cultivation (7 to 12 passages), MSC demonstrated morphological abnormalities, enlargement, attenuated expression of specific surface markers, and ultimately proliferation arrest. Adipogenic differentiation potential decreased whereas the propensity for osteogenic differentiation increased. mRNA expression profiling revealed a consistent pattern of alterations in the global gene expression signature of MSC at different passages. These changes are not restricted to later passages, but are continuously acquired with increasing passages. Genes involved in cell cycle, DNA replication and DNA repair are significantly down-regulated in late passages. Genes from chromosome 4q21 were over-represented among differentially regulated transcripts. Differential expression of 10 genes has been verified in independent donor samples as well as in MSC that were isolated under different culture conditions. Furthermore, miRNA expression profiling revealed an up-regulation of hsa-mir-371, hsa-mir-369-5P, hsa-mir-29c, hsa-mir-499 and hsa-let-7f upon in vitro propagation. Our studies indicate that replicative senescence of MSC preparations is a continuous process starting from the first passage onwards. This process includes far reaching alterations in phenotype, differentiation potential, global gene expression patterns, and miRNA profiles that need to be considered for therapeutic application of MSC preparations.

Blood formation is believed to occur through step-wise progression of haematopoietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent progenitors. However, this model is based on the analysis of predefined flow-sorted cell populations. Here we integrated flow cytometric, transcriptomic and functional data at single-cell resolution to quantitatively map early differentiation of human HSCs towards lineage commitment. During homeostasis, individual HSCs gradually acquire lineage biases along multiple directions without passing through discrete hierarchically organized progenitor populations. Instead, unilineage-restricted cells emerge directly from a “Continuum of LOw primed UnDifferentiated hematopoietic stem- and progenitor cells” (CLOUD-HSPCs). Distinct gene expression modules operate in a combinatorial manner to control stemness, early lineage priming and the subsequent progression into all major branches of haematopoiesis. These data reveal a continuous landscape of human steady state haematopoiesis downstream of HSCs and provide a basis for the understanding of hematopoietic malignancies.

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