Abhishek Sohni

Mesenchymal Stem Cells Migration Homing and Tracking

In this review, we discuss the migration and homing ability of mesenchymal stem cells (MSCs) and MSC-like cells and factors influencing this. We also discuss studies related to the mechanism of migration and homing and the approaches undertaken to enhance it. Finally, we describe the different methods available and frequently used to track and identify the injected cells in vivo.

Differentiation Potential of Human Postnatal Mesenchymal Stem Cells, Mesoangioblasts, and Multipotent Adult Progenitor Cells Reflected in Their Transcriptome and Partially Influenced by the Culture Conditions†‡§

Several adherent postnatal stem cells have been described with different phenotypic and functional properties. As many of these cells are being considered for clinical therapies, it is of great importance that the identity and potency of these products is validated. We compared the phenotype and functional characteristics of human mesenchymal stem cells (hMSCs), human mesoangioblasts (hMab), and human multipotent adult progenitor cells (hMAPCs) using uniform standardized methods. Human MAPCs could be expanded significantly longer in culture. Differences in cell surface marker expression were found among the three cell populations with CD140b being a distinctive marker among the three cell types. Differentiation capacity towards adipocytes, osteoblasts, chondrocytes, and smooth muscle cells in vitro, using established protocols, was similar among the three cell types. However, only hMab differentiated to skeletal myocytes, while only hMAPCs differentiated to endothelium in vitro and in vivo. A comparative transcriptome analysis confirmed that the three cell populations are distinct and revealed gene signatures that correlated with their specific functional properties. Furthermore, we assessed whether the phenotypic, functional, and transcriptome features were mediated by the culture conditions. Human MSCs and hMab cultured under MAPC conditions became capable of generating endothelial‐like cells, whereas hMab lost some of their ability to generate myotubes. By contrast, hMAPCs cultured under MSC conditions lost their endothelial differentiation capacity, whereas this was retained when cultured under Mab conditions, however, myogenic capacity was not gained under Mab conditions. These studies demonstrate that hMSCs, hMab, and hMAPCs have different properties that are partially mediated by the culture conditions. STEM CELLS 2011;29:871–882

Multipotent Adult Progenitor Cells

We here discuss the potency and characteristics of various adult derived adherent stem cells with special focus on multipotent adult progenitor cells (MAPC) isolated first in 2002 in our lab. We describe the potency of MAPC, our current understanding in relationship with novel insights gained in epigenetic modifications that increase cellular potency, and their possible clinical applications.

18F-FDG Labeling of Mesenchymal Stem Cells and Multipotent Adult Progenitor Cells for PET Imaging: Effects on Ultrastructure and Differentiation Capacity

Because of their extended differentiation capacity, stem cells have gained great interest in the field of regenerative medicine. For the development of therapeutic strategies, more knowledge on the in vivo fate of these cells has to be acquired. Therefore, stem cells can be labeled with radioactive tracer molecules such as 18F-FDG, a positron-emitting glucose analog that is taken up and metabolically trapped by the cells. The aim of this study was to optimize the radioactive labeling of mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) in vitro with 18F-FDG and to investigate the potential radiotoxic effects of this labeling procedure with a range of techniques, including transmission electron microscopy (TEM). Methods: Mouse MSCs and rat MAPCs were used for 18F-FDG uptake kinetics and tracer retention studies. Cell metabolic activity, proliferation, differentiation and ultrastructural changes after labeling were evaluated using an Alamar Blue reagent, doubling time calculations and quantitative TEM, respectively. Additionally, mice were injected with MSCs and MAPCs prelabeled with 18F-FDG, and stem cell biodistribution was investigated using small-animal PET. Results: The optimal incubation period for 18F-FDG uptake was 60 min. Significant early tracer washout was observed, with approximately 30%–40% of the tracer being retained inside the cells 3 h after labeling. Cell viability, proliferation, and differentiation capacity were not severely affected by 18F-FDG labeling. No major changes at the ultrastructural level, considering mitochondrial length, lysosome size, the number of lysosomes, the number of vacuoles, and the average rough endoplasmic reticulum width, were observed with TEM. Small-animal PET experiments with radiolabeled MAPCs and MSCs injected intravenously in mice showed a predominant accumulation in the lungs and a substantial elution of 18F-FDG from the cells. Conclusion: MSCs and MAPCs can be successfully labeled with 18F-FDG for molecular imaging purposes. The main cellular properties are not rigorously affected. TEM confirmed that the cells’ ultrastructural properties are not influenced by 18F-FDG labeling. Small-animal PET studies confirmed the intracellular location of the tracer and the possibility of imaging injected prelabeled stem cell types in vivo. Therefore, direct labeling of MSCs and MAPCs with 18F-FDG is a suitable technique to noninvasively assess cell delivery and early retention with PET.

Dynamic Switching of Active Promoter and Enhancer Domains Regulates Tet1 and Tet2 Expression during Cell State Transitions between Pluripotency and Differentiation

ABSTRACT The Tet 5-methylcytosine dioxygenases catalyze DNA demethylation by producing 5-hydroxymethylcytosine and further oxidized products. Tet1 and Tet2 are highly expressed in mouse pluripotent cells and downregulated to different extents in somatic cells, but the transcriptional mechanisms are unclear. Here we defined the promoter and enhancer domains in Tet1 and Tet2. Within a 15-kb “superenhancer” of Tet1, there are two transcription start sites (TSSs) with different activation patterns during development. A 6-kb promoter region upstream of the distal TSS is highly active in naive pluripotent cells, autonomously reports Tet1 expression in a transgenic system, and rapidly undergoes DNA methylation and silencing upon differentiation in cultured cells and native epiblast. A second TSS downstream, associated with a constitutively weak CpG-rich promoter, is activated by a neighboring enhancer in naive embryonic stem cells (ESCs) and primed epiblast-like cells (EpiLCs). Tet2 has a CpG island promoter with pluripotency-independent activity and an ESC-specific distal intragenic enhancer; the latter is rapidly downregulated in EpiLCs. Our study reveals distinct modes of transcriptional regulation at Tet1 and Tet2 during cell state transitions of early development. New transgenic reporters using Tet1 and Tet2 cis-regulatory domains may serve to distinguish nuanced changes in pluripotent states and the underlying epigenetic variations.

Novel Analysis of Clonal Diversification in Blood B Cell and Bone Marrow Plasma Cell Clones in Immunoglobulin Light Chain Amyloidosis

Immunoglobulin light chain amyloidosis (AL) is characterized by a limited clonal expansion of plasma cells and amyloid formation. Here, we report restriction in the diversity of VL gene usage with a dominance of clonally related B cells in the peripheral blood (PB) isotype-specific repertoire of AL patients. A rigorous quantification of lineage trees reveals presence of intraclonal variations in the PB clones compared to the bone marrow (BM) clones, which suggests a common precursor that is still subject to somatic mutation. When compared to normal BM and PB B cells, AL clones showed significant but incomplete impairment of antigenic selection, which could not be detected by conventional R and S mutation analysis. Therefore, graphical analysis of B cell lineage trees and mathematical quantification of tree properties provide novel insights into the process of B cell clonal evolution in AL.

MAPC culture conditions support the derivation of cells with nascent hypoblast features from bone marrow and blastocysts

Dear Editor, We previously demonstrated (Jiang et al., 2002) that rodent multipotent adult progenitor cells (MAPC) can self-renew longterm while maintaining multilineage differentiation capacity. Rodent MAPC express a number of pluripotency-related transcription factors (TF) including Oct4 and Rex1 but not Nanog and Sox2, two other TF known to play a significant role in the maintenance of the pluripotency of embryonic stem cells (ESC) (Ulloa-Montoya et al., 2007). However, rodent MAPC express several TF, including Gata4, Gata6, Sox7 and Sox17, typically expressed in the nascent hypoblast of the developing inner cell mass (ICM) (Nichols and Smith, 2011) and in the recently described rat extrambryonic endodermal precursor cells (rXEN-P), which are isolated from blastocyst (Debeb et al., 2009). We derived in 4/12 independent isolations one or more rMAPC lines, by culturing rat BM cells in rMAPC medium (rMAPC isolation scheme, Supplementary Figure S1). After 4 weeks of culture, BM cells were depleted of CD45+ cells and 2–8 weeks later, clusters of refractile and small cells appeared, which became the preponderant cell type within 10 days (Figure 1A). Nearly all cells from the established lines expressed Oct4, Gata4, Gata6, Sox7 and Sox17 transcripts and proteins (Figure 1B and Supplementary Figure S2A and B), as well the surface markers SSEA1 and CD31 (Figure 1C and Supplementary Figure S2C), both markers of the early ICM. Although rMAPC lines express Oct4, previous studies (Lengner et al., 2007) demonstrated that Oct4+ cells cannot be detected in adult mouse tissues and that Oct4 is not required for postnatal tissue homeostasis. Based on Lengner’s findings, we hypothesized that the rMAPC phenotype could be the result of a culture-induced reprogramming. We therefore analysed BM-cultures during 2 independent rMAPC isolations before, during and after the appearance of the refractile and small cells. We could not identify any Oct4+ or SSEA1+/CD31+ cells in more than one million cells analyzed after CD45+ cells depletion (Figure 1C and D, and Supplementary Figure S2D and E), several weeks before the appearance of the refractile cells positive for these markers. RT-qPCR analysis further demonstrated that acquisition of the typical rMAPC morphology was associated with .1000fold increase in expression of Oct4 and the typical hypoblast gene transcripts (Figure 1E and Supplementary Figure S2F). Although some rMAPC lines had karyotypical abnormalities (Supplementary Figure S2G and Table S1), some lines did not, suggesting that the rMAPC phenotype is not induced by a specific translocation, duplication and/or deletion. These studies demonstrate that rMAPC do not exist in BM and that this hypoblast phenotype is acquired upon prolonged in vitro culture. rMAPC may represent a rare event of in vitro reprogramming, resembling what has been observed during spermatogonial stem cell (Guan et al., 2006; Kanatsu-Shinohara et al., 2008; Ko et al., 2009) and epiblast stem cell (Bao et al., 2009) de-differentiation to ESC-like cells, when cultured under ESC conditions. Because the gene expression pattern of rMAPC (Ulloa-Montoya et al., 2007) and rXEN-P cells (Debeb et al., 2009) is highly similar, we next asked whether BM cells were reprogrammed to a hypoblast/extraembryonic progenitor fate. To investigate this, we tested whether rMAPC could be cultured under rXEN-P conditions and vice versa. When established rXEN-P lines were cultured under rMAPC conditions for 1–2 passages, they grew dispersed, acquiring the typical rMAPC morphology (Supplementary Figure S3A). rXEN-P cells became homogeneously Oct4+/Gata4+ and the percentage of SSEA1+ cells increased (Supplementary Figure S3B and E). RT-qPCR revealed that no differences in RNA expression for hypoblast genes could be detected in XEN-P lines, once cultured in MAPC conditions, except for higher levels of Sox17 and lower levels of Tmprss2 (Supplementary Figure S3D). By contrast, when rMAPC were cultured in XEN-P medium on rat embryonic feeders, typical XEN-P colonies were generated, i.e. Oct42/Gata4+ epithelioid cells with a rim of loosely attached small refractile cells that are Oct4+/Gata4+ (Supplementary Figure S3F and G). Moreover, the percentage of SSEA1+ cells decreased (Supplementary Figure S3J); consistently, RT-qPCR revealed a decrease in Sox17 and an increase in Tmprss2 (Supplementary Figure S3I). Cell doubling time of rMAPC or rXEN-P cells cultured in MAPC conditions was slightly faster than in XEN-P conditions (Supplementary Figure S3A and F). Therefore, rMAPC culture conditions supported the feederfree growth of established XEN-P clones in a more homogenous and immature state. To further define the relationship between rMAPC, XEN-P and typical XEN cells, rMAPC and rXEN-P cells were also cultured under standard XEN conditions (Kunath et al., 2005) without exogenous LIF (Supplementary Figure S4A). rXEN-P and rMAPC cells in XEN conditions, formed extraembryonic endodermal colonies with significantly lower proliferation rate (Supplementary Figure S4B and E). Expression of hypoblast gene transcripts doi:10.1093/jmcb/mjs046 Journal of Molecular Cell Biology (2012), 4, 423–426 | 423 Published online August 9, 2012

Lineage-specific functions of TET1 in the postimplantation mouse embryo

The mammalian TET enzymes catalyze DNA demethylation. While they have been intensely studied as major epigenetic regulators, little is known about their physiological roles and the extent of functional redundancy following embryo implantation. Here we define non-redundant roles for TET1 at an early postimplantation stage of the mouse embryo, when its paralogs Tet2 and Tet3 are not detectably expressed. TET1 regulates numerous genes defining differentiation programs in the epiblast and extraembryonic ectoderm. In epiblast cells, TET1 demethylates gene promoters via hydroxymethylation and maintains telomere stability. Surprisingly, TET1 represses a majority of epiblast target genes independently of methylation changes, in part through regulation of the gene encoding the transcriptional repressor JMJD8. Dysregulated gene expression in the absence of TET1 causes embryonic defects, which are partially penetrant in an inbred strain but fully lethal in non-inbred mice. Collectively, our study highlights an interplay between the catalytic and non-catalytic activities of TET1 that is essential for normal development.

TGFβ1-induced Baf60c regulates both smooth muscle cell commitment and quiescence.

Smooth muscle cells (SMCs) play critical roles in a number of diseases; however, the molecular mechanism underlying their development is unclear. Although the role of TGFβ1 signaling in SMC development is well established, the downstream molecular signals are not fully understood. We used several rat multipotent adult progenitor cell ((r)MAPC) lines that express levels of Oct4 mRNA similar to hypoblast stem cells (HypoSC), and can differentiate robustly to mesodermal and endodermal cell types. TGFβ1 alone, or with PDGF-BB, induces differentiation of rMAPCs to SMCs, which expressed structural SMC proteins, including α-smooth muscle actin (αSMA), and contribute to the SMC coat of blood vessels in vivo. A genome-wide time-course transcriptome analysis revealed that transcripts of Baf60c, part of the SWI/SNF actin binding chromatin remodeling complex D-3 (SMARCD3/BAF60c), were significantly induced during MAPC-SMC differentiation. We demonstrated that BAF60c is a necessary co-regulator of TGFβ1 mediated induction of SMC genes. Knock-down of Baf60c decreased SMC gene expression in rMAPCs whereas ectopic expression of Baf60c was sufficient to commit rMAPCs to SMCs in the absence of exogenous cytokines. TGFβ1 activates Baf60c via the direct binding of SMAD2/3 complexes to the Baf60c promoter region. Chromatin- and co-immunoprecipitation studies demonstrated that regulation of SMC genes by BAF60c is mediated via interaction with SRF binding CArG box-containing promoter elements in SMC genes. We noted that compared with TGFβ1, Baf60c overexpression in rMAPC yielded SMC with a more immature phenotype. Similarly, Baf60c induced an immature phenotype in rat aortic SMCs marked by increased cell proliferation and decreased contractile marker expression. Thus, Baf60c is important for TGFβ-mediated commitment of primitive stem cells (rMAPCs) to SMCs and is associated with induction of a proliferative state of quiescent SMCs. The MAPC-SMC differentiation system may be useful for identification of additional critical (co-)regulators of SMC development.