Gene Expression Profile and Functionality of ESC-Derived Lin-ckit+Sca-1+ Cells Are Distinct from Lin-ckit+Sca-1+ Cells Isolated from Fetal Liver or Bone Marrow

In vitro bioreactor-based cultures are being extensively investigated for large-scale production of differentiated cells from embryonic stem cells (ESCs). However, it is unclear whether in vitro ESC-derived progenitors have similar gene expression profiles and functionalities as their in vivo counterparts. This is crucial in establishing the validity of ESC-derived cells as replacements for adult-isolated cells for clinical therapies. In this study, we compared the gene expression profiles of Lin-ckit+Sca-1+ (LKS) cells generated in vitro from mouse ESCs using either static or bioreactor-based cultures, with that of native LKS cells isolated from mouse fetal liver (FL) or bone marrow (BM). We found that in vitro-generated LKS cells were more similar to FL- than to BM LKS cells in gene expression. Further, when compared to cells derived from bioreactor cultures, static culture-derived LKS cells showed fewer differentially expressed genes relative to both in vivo LKS populations. Overall, the expression of hematopoietic genes was lower in ESC-derived LKS cells compared to cells from BM and FL, while the levels of non-hematopoietic genes were up-regulated. In order to determine if these molecular profiles correlated with functionality, we evaluated ESC-derived LKS cells for in vitro hematopoietic-differentiation and colony formation (CFU assay). Although static culture-generated cells failed to form any colonies, they did differentiate into CD11c+ and B220+ cells indicating some hematopoietic potential. In contrast, bioreactor-derived LKS cells, when differentiated under the same conditions failed to produce any B220+ or CD11c+ cells and did not form colonies, indicating that these cells are not hematopoietic progenitors. We conclude that in vitro culture conditions significantly affect the transcriptome and functionality of ESC-derived LKS cells and although in vitro differentiated LKS cells were lineage negative and expressed both ckit and Sca-1, these cells, especially those obtained from dynamic cultures, are significantly different from native cells of the same phenotype.This work was partially supported through National Institutes of Health grant number EB005026 to KR and PWT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.Biomedical Engineerin

Three-dimensional multipotent progenitor cell aggregates for expansion, osteogenic differentiation and ‘in vivo’ tracing with AAV vector serotype 6

Multipotent adult progenitor cells (MAPC) are bone marrow-derived stem cells with a high growth rate suitable for therapeutical applications as three-dimensional (3D) aggregates. Combined applications of osteogenically differentiated MAPC (OD-MAPC) aggregates and adeno-associated viral vectors (AAV) in bone bioengineering are still deferred until information regarding expansion technologies, osteogenic potential, and AAV cytotoxicity and transduction efficiency is better understood. In this study, we tested whether self-complementary AAV (scAAV) can potentially be used as a gene delivery system in a OD-MAPC-based “in vivo” bone formation model in the craniofacial region. Both expansion of rat MAPC (rMAPC) and osteogenic differentiation with dexamethasone were also tested in 3D aggregate culture systems “in vitro” and “vivo”. Rat MAPCs (rMAPCs) grew as undifferentiated aggregates for 4 days with a population doubling time of 37h. After expansion, constant levels of Oct4 transcripts, and Oct4 and CD31 surface markers were observed, which constitute a hallmark of rMAPCs undifferentiated stage. Dexamethasone effectively mediated rMAPC osteogenic differentiation by inducing the formation of a mineralized collagen type I network, and facilitated the activation of the wnt/β-catenin, a crucial pathway in skeletal development. To investigate the genetic modification of rMAPCs grown as 3D aggregates prior to implantation, scAAV serotypes 2, 3, and 6 were evaluated. scAAV6 packaged with the enhanced green fluorescent protein expression cassette efficiently mediated long-term transduction (10 days) “in vitro” and “vivo”. The reporter transduction event allowed the tracing of OD-rMAPC (induced by dexamethasone) aggregates following OD-rMAPC transfer into a macro-porous hydroxyapatite scaffold implanted in a rat calvaria model. Furthermore, the scAAV6-transduced OD-rMAPC generated a bone-like matrix with a collagenous matrix rich in bone specific proteins (osteocalcin and osteopontin) in the scaffold macro-pores 10 days post-implantation. Newly formed bone was also observed in the interface between native bone and scaffold. The collective work supports future bone tissue engineering applications of 3D MAPC cultures for expansion, bone formation, and the ability to genetically alter these cells using scAAV vectors