Yu-Jen Chang

Functional Network Analysis of the Transcriptomes of Mesenchymal Stem Cells Derived from Amniotic Fluid, Amniotic Membrane, Cord Blood, and Bone Marrow

Using high‐density oligonucleotide microarrays and functional network analyses, we examined whether MSCs derived from four different origins exhibited unique gene expression profiles individually and then compared the gene expression profiles of all MSCs with those of fetal organs. Our results indicated that within each group of MSCs from the same origin, the variability of the gene expression levels was smaller than that between groups of different origins. Functional genomic studies revealed the specific roles of MSCs from different origins. Our results suggest that amniotic fluid MSCs may initiate interactions with the uterus by upregulating oxytocin and thrombin receptors. Amniotic membrane MSCs may play a role in maintaining homeostasis of fluid and electrolytes by regulating the networks of endothelin, neprilysin, bradykinin receptors, and atrial natriuretic peptide. Cord blood MSCs may be involved in innate immune systems as the neonatal defense system against the earliest encountered pathogens. Adult bone marrow MSCs may be an important source not only of all blood lineages but also of bone formation. However, in spite of the different gene expression profiles seen in MSCs derived from different origins, a set of core gene expression profiles was preserved in these four kinds of MSCs. The core signature transcriptomes of all MSCs, when contrasted against those of fetal organs, included genes involved in the regulation of extracellular matrix and adhesion, transforming growth factor‐β receptor signaling, and the Wnt signaling pathways.

Disparate Mesenchyme-Lineage Tendencies in Mesenchymal Stem Cells from Human Bone Marrow and Umbilical Cord Blood

Bone marrow and umbilical cord blood are reported to be the main sources of mesenchymal stem cells (MSCs), which have been proposed for many clinical applications. This study evaluated and quantitated the differentiation potential of bone marrow–derived MSCs (bmMSCs) and cord blood–derived MSCs (cbMSCs) by in vitro induction. Results indicated that cbMSCs had a significantly stronger osteogenic potential but lower capacity for adipogenic differentiation than bmMSCs. Leptin, an important regulator of mesenchymal differentiation, has a significantly stronger effect of promoting osteogenesis and inhibiting adipogenesis in bmMSCs than in cbMSCs. Moreover, Cbfa1 mRNA expression in bmMSCs and cbMSCs was affected to different degrees by leptin during osteogenesis. In contrast, leptin reduced PPARγ2 mRNA expression to the same level during adipogenesis in both types of MSCs. These results demonstrate the disparate capacities of MSCs from bone marrow and cord blood and suggest that they be used differently in experimental and therapeutic studies. In addition, the disparate differentiation tendencies of MSCs from different sources should be considered in further applications.

Characterization of two populations of mesenchymal progenitor cells in umbilical cord blood

Umbilical cord blood (UCB) is a valuable source for hematopoietic progenitor cell therapy. Moreover, it contains another subset of non‐hematopoietic population referred to as mesenchymal progenitor cells (MPCs), which can be ex vivo expanded and differentiated into osteoblasts, chondrocytes and adipocytes. In this study, we successfully isolated the clonogenic MPCs from UCB by limiting dilution method. These cells exhibited two different morphologic phenotypes, including flattened fibroblasts (majority) and spindle‐shaped fibroblasts (minority). Both types of MPCs shared similar cell surface markers except CD90 and had similar osteogenic and chondrogenic potentials. However, the spindle‐shaped clones possessed the positive CD90 expression and showed a greater tendency in adipogenesis, while the flattened clones were CD90 negative cells and showed a lower tendency in adipogenesis. The high number of flattened MPCs might be linked to the less sensitivity of UCB‐derived MPCs in adipogenic differentiation.

Isolation of Mesenchymal Stem Cells with Neurogenic Potential from the Mesoderm of the Amniotic Membrane

The amniotic membrane has been clinically applied as a therapeutic material in wound covering and corneal surface reconstruction. Recently, mesenchymal stem cells (MSCs) have been isolated from the placenta, specifically from the amniotic membrane. However, the localization of MSCs in the amniotic membrane has not been determined. In this study, term placenta was collected, and we performed immunohistochemical staining techniques to identify and localize MSCs in the mesoderm of the amniotic membrane in situ with MSC antibodies, including CD90 and CD105. We further directly cultured and characterized MSCs from the amniotic membrane mesoderm (AMSCs). The AMSCs were easily isolated and represented a homogenous fibroblastic morphology at early passages. In addition to MSC surface markers, AMSCs expressed Sox2, Oct-4 and Nanog. AMSCs could be induced into osteocytes, adipocytes and chondrocytes in vitro and show immunosuppressive effects on T-cell proliferation. Under appropriate conditions, AMSCs could differentiate into neuronal-like cells, which were identified by neuronal-specific markers and their ability to secrete dopamine. This study reveals that AMSCs provide a promising source for stem cell studies and also extend the clinical potential of the amniotic membrane in the field of regenerative medicine.

Growth rate regulation of lac operon expression in Escherichia coli is cyclic AMP dependent

In contrast to the ribosomal RNA gene expression increasing with growth rate, transcription of the lac operon is downregulated by cell growth rate. In continuous culture, growth rate regulation of lac promoter was independent of carbon substrate used and its location on the chromosome. Since the lac operon is activated by cyclic adenosine monophosphate (cAMP), which decreases with increasing cell growth rate, expression of plac‐lacZ reporter fusion was analyzed in cya mutant under various growth conditions. The results demonstrated that expression of plac‐lacZ in cya mutant was both lower and growth rate independent. In addition, ppGpp (guanosine tetraphosphate) was not involved in the mechanism of growth rate regulation of the lac promoter. Thus, the results of this study indicate that cAMP mediates the growth rate‐dependent regulation of lac operon expression in Escherichia coli.

SOX9 as a Predictor for Neurogenesis Potentiality of Amniotic Fluid Stem Cells

Preclinical studies of amniotic fluid‐derived cell therapy have been successful in the research of neurodegenerative diseases, peripheral nerve injury, spinal cord injury, and brain ischemia. Transplantation of human amniotic fluid stem cells (AFSCs) into rat brain ventricles has shown improvement in symptoms of Parkinsons disease and also highlighted the minimal immune rejection risk of AFSCs, even between species. Although AFSCs appeared to be a promising resource for cell‐based regenerative therapy, AFSCs contain a heterogeneous pool of distinct cell types, rendering each preparation of AFSCs unique. Identification of predictive markers for neuron‐prone AFSCs is necessary before such stem cell‐based therapeutics can become a reality. In an attempt to identify markers of AFSCs to predict their ability for neurogenesis, we performed a two‐phase study. In the discovery phase of 23 AFSCs, we tested ZNF521/Zfp521, OCT6, SOX1, SOX2, SOX3, and SOX9 as predictive markers of AFSCs for neural differentiation. In the validation phase, the efficacy of these predictive markers was tested in independent sets of 18 AFSCs and 14 dental pulp stem cells (DPSCs). We found that high expression of SOX9 in AFSCs is associated with good neurogenetic ability, and these positive correlations were confirmed in independent sets of AFSCs and DPSCs. Furthermore, knockdown of SOX9 in AFSCs inhibited their neuronal differentiation. In conclusion, the discovery of SOX9 as a predictive marker for neuron‐prone AFSCs could expedite the selection of useful clones for regenerative medicine, in particular, in neurological diseases and injuries.

Recovery of CD45−/Lin−/SSEA-4+ very small embryonic-like stem cells by cord blood bank standard operating procedures

BACKGROUND AIMS Very small embryonic-like (VSEL) stem cells are a rare cell population present in bone marrow, cord blood and other tissues that displays a distinct small cell size and the ability to give rise to cells of the three germ layers. VSEL stem cells were reported to be discarded in the red blood cell fraction by Ficoll-Paque density gradient centrifugation during the processing of bone marrow and cord blood specimens. However, most cord blood banks do not include density gradient centrifugation in their procedures while red blood cells are removed by Hespan sedimentation following the Cord Blood Transplantation Study cord blood bank standard operating procedures (COBLT SOP). To clarify the retention of VSEL stem cells, we investigated the recovery of VSEL stem cells following COBLT SOP guidelines. METHODS The recovery of CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells of umbilical cord blood was examined by flow cytometry before and after COBLT SOP processing, and relative expression of pluripotent genes was analyzed by quantitative polymerase chain reaction. RESULTS CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells were mostly recovered in the final products following COBLT SOP guidelines. The expression of pluripotent genes could be maintained at >80% in products after hetastarch (Hespan; B. Braun Medical Inc., Irvine, CA, USA) processing. CONCLUSIONS The rare sub-population of CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells survived after Hespan sedimentation. This finding suggests that umbilical cord blood units cryopreserved by COBLT SOP in cord blood banks should retain most VSEL stem cells present in the un-processed specimens.