Loss of O-GlcNAc transferase in neural stem cells impairs corticogenesis.

The proper development of the cerebral cortex is essential for brain formation and functioning. O-GlcNAcylation, an important posttranslational modification, regulates the pathways critical for neuronal health and the survival of the cerebral cortex in neurodegenerative diseases. However, the role of O-GlcNAcylation in regulating cerebral cortical development at the embryonic and early postnatal (0-21 days) stages is still largely unknown. Here we report that the selective deletion of O-GlcNAc transferase (OGT) in neural stem cells (NSCs) in mice led to a series of severe brain developmental deficits, including dramatic shrinkage of cortical and hippocampal histoarchitecture, widespread neuronal apoptosis, decrease in cell proliferation, induction of endoplasmic reticulum (ER) stress, and inhibition of neuronal dendritic and axonal differentiation. The pathology of corticogenesis deficits caused by OGT deletion may largely rely on complicated biological processes, such as proliferation, apoptosis and differentiation. Our results suggest that dysfunctional O-GlcNAcylation in NSCs may be an important contributor to neurodevelopmental diseases.

Wnt5a suppresses osteoblastic differentiation of human periodontal ligament stem cell-like cells via Ror2/JNK signaling.

Wnt5a, a non-canonical Wnt protein, is known to play important roles in several cell functions. However, little is known about the effects of Wnt5a on osteoblastic differentiation of periodontal ligament (PDL) cells. Here, we examined the effects of Wnt5a on osteoblastic differentiation and associated intracellular signaling in human PDL stem/progenitor cells (HPDLSCs). We found that Wnt5a suppressed expression of bone-related genes (ALP, BSP, and Osterix) and alizarin red-positive mineralized nodule formation in HPDLSCs under osteogenic conditions. Immunohistochemical analysis revealed that a Wnt5a-related receptor, receptor tyrosine kinase-like orphan receptor 2 (Ror2), was expressed in rat PDL tissue. Interestingly, knockdown of Ror2 by siRNA inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Moreover, Western blotting analysis showed that phosphorylation of the intracellular signaling molecule, c-Jun N-terminal kinase (JNK) was upregulated in HPDLSCs cultured in osteoblast induction medium with Wnt5a, but knockdown of Ror2 by siRNA downregulated the phosphorylation of JNK. We also examined the effects of JNK inhibition on Wnt5a-induced suppression of osteoblastic differentiation of HPDLSCs. The JNK inhibitor, SP600125 inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Additionally, SP600125 inhibited the Wnt5a-induced suppression of the alizarin red-positive reaction in HPDLSCs. These results suggest that Wnt5a suppressed osteoblastic differentiation of HPDLSCs through Ror2/JNK signaling. Non-canonical Wnt signaling, including Wnt5a/Ror2/JNK signaling, may function as a negative regulator of mineralization, preventing the development of non-physiological mineralization in PDL tissue.

Querkopf is a key marker of self-renewal and multipotency of adult neural stem cells.

Adult neural stem cells (NSCs) reside in the subventricular zone (SVZ) and produce neurons throughout life. Although their regenerative potential has kindled much interest, few factors regulating NSCs in vivo are known. Among these is the histone acetyltransferase querkopf (QKF, also known as MYST4, MORF, KAT6B), which is strongly expressed in a small subset of cells in the neurogenic subventricular zone. However, the relationship between Qkf gene expression and the hierarchical levels within the neurogenic lineage is currently unknown. We show here that the 10% of SVZ cells with the highest Qkf expression possess the defining NSC characteristics of multipotency and self-renewal and express markers previously shown to enrich for NSCs. A fraction of cells expressing Qkf at medium to high levels is enriched for multipotent progenitor cells with limited self-renewal, followed by a population containing migrating neuroblasts. Cells low in Qkf promoter activity are predominantly ependymal cells. In addition, we show that mice deficient for Bmi1, a central regulator of NSC self-renewal, show an age-dependent decrease in the strongest Qkf-expressing cell population in the SVZ. Our results show a strong relationship between Qkf promoter activity and stem cell characteristics, and a progressive decrease in Qkf gene activity as lineage commitment and differentiation proceed in vivo.

Pharmaceutical modulation of canonical Wnt signaling in multipotent stromal cells for improved osteoinductive therapy.

Human mesenchymal stem cells (hMSCs) from bone marrow are regarded as putative osteoblast progenitors in vivo and differentiate into osteoblasts in vitro. Positive signaling by the canonical wingless (Wnt) pathway is critical for the differentiation of MSCs into osteoblasts. In contrast, activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma)-mediated pathway results in adipogenesis. We therefore compared the effect of glycogen-synthetase-kinase-3beta (GSK3beta) inhibitors and PPARgamma inhibitors on osteogenesis by hMSCs. Both compounds altered the intracellular distribution of beta-catenin and GSK3beta in a manner consistent with activation of Wnt signaling. With osteogenic supplements, the GSK3beta inhibitor 6-bromo-indirubin-3'-oxime (BIO) and the PPARgamma inhibitor GW9662 (GW) enhanced early osteogenic markers, alkaline phosphatase (ALP), and osteoprotegerin (OPG) by hMSCs and transcriptome analysis demonstrated up-regulation of genes encoding bone-related structural proteins. At higher doses of the inhibitors, ALP levels were attenuated, but dexamethasone-induced biomineralization was accelerated. When hMSCs were pretreated with BIO or GW and implanted into experimentally induced nonself healing calvarial defects, GW treatment substantially increased the capacity of the cells to repair the bone lesion, whereas BIO treatment had no significant effect. Further investigation indicated that unlike GW, BIO induced cell cycle inhibition in vitro. Furthermore, we found that GW treatment significantly reduced expression of chemokines that may exacerbate neutrophil- and macrophage-mediated cell rejection. These data suggest that use of PPARgamma inhibitors during the preparation of hMSCs may enhance the capacity of the cells for osteogenic cytotherapy, whereas adenine analogs such as BIO can adversely affect the viability of hMSC preparations in vitro and in vivo.

Potential role of 20S proteasome in maintaining stem cell integrity of human bone marrow stromal cells in prolonged culture expansion.

Human bone marrow stromal cells (hBMSCs) could be used in clinics as precursors of multiple cell lineages following proper induction. Such application is impeded by their characteristically short lifespan, together with the increasing loss of proliferation capability and progressive reduction of differentiation potential after the prolonged culture expansion. In the current study, we addressed the possible role of 20S proteasomes in this process. Consistent with prior reports, long-term in vitro expansion of hBMSCs decreased cell proliferation and increased replicative senescence, accompanied by reduced activity and expression of the catalytic subunits PSMB5 and PSMB1, and the 20S proteasome overall. Application of the proteasome inhibitor MG132 produced a senescence-like phenotype in early passages, whereas treating late-passage cells with 18α-glycyrrhetinic acid (18α-GA), an agonist of 20S proteasomes, delayed the senescence progress, enhancing the proliferation and recovering the capability of differentiation. The data demonstrate that activation of 20S proteasomes assists in counteracting replicative senescence of hBMSCs expanded in vitro.

Vasa genes: emerging roles in the germ line and in multipotent cells.

Sexually reproducing metazoans establish a cell lineage during development that is ultimately dedicated to gamete production. Work in a variety of animals suggests that a group of conserved molecular determinants act in this germ line maintenance and function. The most universal of these genes are Vasa and Vasa-like DEAD-box RNA helicase genes. However, recent evidence indicates that Vasa genes also function in other cell types, distinct from the germ line. Here we evaluate our current understanding of Vasa function and its regulation during development, addressing Vasa's emerging role in multipotent cells. We also explore the evolutionary diversification of the N-terminal domain of this gene and how this impacts the association of Vasa with nuage-like perinuclear structures.

Leukocyte tyrosine kinase functions in pigment cell development.

A fundamental problem in developmental biology concerns how multipotent precursors choose specific fates. Neural crest cells (NCCs) are multipotent, yet the mechanisms driving specific fate choices remain incompletely understood. Sox10 is required for specification of neural cells and melanocytes from NCCs. Like sox10 mutants, zebrafish shady mutants lack iridophores; we have proposed that sox10 and shady are required for iridophore specification from NCCs. We show using diverse approaches that shady encodes zebrafish leukocyte tyrosine kinase (Ltk). Cell transplantation studies show that Ltk acts cell-autonomously within the iridophore lineage. Consistent with this, ltk is expressed in a subset of NCCs, before becoming restricted to the iridophore lineage. Marker analysis reveals a primary defect in iridophore specification in ltk mutants. We saw no evidence for a fate-shift of neural crest cells into other pigment cell fates and some NCCs were subsequently lost by apoptosis. These features are also characteristic of the neural crest cell phenotype in sox10 mutants, leading us to examine iridophores in sox10 mutants. As expected, sox10 mutants largely lacked iridophore markers at late stages. In addition, sox10 mutants unexpectedly showed more ltk-expressing cells than wild-type siblings. These cells remained in a premigratory position and expressed sox10 but not the earliest neural crest markers and may represent multipotent, but partially-restricted, progenitors. In summary, we have discovered a novel signalling pathway in NCC development and demonstrate fate specification of iridophores as the first identified role for Ltk.

The ERK5 and ERK1/2 signaling pathways play opposing regulatory roles during chondrogenesis of adult human bone marrow-derived multipotent progenitor cells.

Adult human bone marrow-derived multipotent progenitor cells (MPCs) are able to differentiate into a variety of specialized cell types, including chondrocytes, and are considered a promising candidate cell source for use in cartilage tissue engineering. In this study, we examined the regulation of MPC chondrogenesis by mitogen-activated protein kinases in an attempt to better understand how to generate hyaline cartilage in the laboratory that more closely resembles native tissue. Specifically, we employed the high-density pellet culture model system to assess the roles of ERK5 and ERK1/2 pathway signaling in MPC chondrogenesis. Western blotting revealed that high levels of ERK5 phosphorylation correlate with low levels of MPC chondrogenesis and that as TGF-beta 3-enhanced MPC chondrogenesis proceeds, phospho-ERK5 levels steadily decline. Conversely, levels of phospho-ERK1/2 paralleled the progression of MPC chondrogenesis. siRNA-mediated knockdown of ERK5 pathway components MEK5 and ERK5 resulted in increased MPC pellet mRNA transcript levels of the cartilage-characteristic marker genes SOX9, COL2A1, AGC, L-SOX5, and SOX6, as well as enhanced accumulation of SOX9 protein, collagen type II protein, and Alcian blue-stainable proteoglycan. In contrast, knockdown of ERK1/2 pathway members MEK1 and ERK1 decreased expression of all chondrogenic markers tested. Finally, overexpression of MEK5 and ERK5 also depressed MPC chondrogenesis, as indicated by diminished activity of a co-transfected collagen II promoter-luciferase reporter construct. In conclusion, our results suggest a novel role for the ERK5 pathway as an important negative regulator of adult human MPC chondrogenesis and illustrate that the ERK5 and ERK1/2 kinase cascades play opposing roles regulating MPC cartilage formation.

Neonatal mouse testis-derived multipotent germline stem cells improve the cardiac function of acute ischemic heart mouse model.

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. We previously reported that undifferentiated mGS cells are phenotypically similar to embryonic stem cells and that fetal liver kinase 1 (Flk1)(+) mGS cells have a similar potential to differentiate into cardiomyocytes and endothelial cells compared with Flk1(+) embryonic stem cells. Here, we transplanted these Flk1(+) mGS cells into an ischemic heart failure mouse model to evaluate the improvement in cardiac function. Significant increase in left ventricular wall thickness of the infarct area, left ventricular ejection fraction and left ventricular maximum systolic velocity was observed 4weeks after when sorted Flk1(+) mGS cells were transplanted directly into the hearts of the acute ischemic model mice. Although the number of cardiomyocytes derived from Flk1(+) mGS cells were too small to account for the improvement in cardiac function but angiogenesis around ischemic area was enhanced in the Flk1(+) mGS cells transplanted group than the control group and senescence was also remarkably diminished in the early phase of ischemia according to ß-galactosidase staining assay. In conclusion, Flk1(+) mGS cell transplantation can improve the cardiac function of ischemic hearts by promoting angiogenesis and by delaying host cell death via senescence.

Sustained epidermal growth factor receptor levels and activation by tethered ligand binding enhances osteogenic differentiation of multi-potent marrow stromal cells.

Epidermal growth factor receptor (EGFR)-mediated signaling helps regulate bone development and healing through its effects on osteogenic cells. Here, we show how EGFR activity and osteogenic differentiation responses in primary human bone marrow-derived multipotent stromal cells (MSCs) are influenced by presenting covalently tethered epidermal growth factor (tEGF) on the culture substratum, a presentation mode that reduces EGFR internalization and restricts signaling to the cell surface. In both absence and presence of tEGF, MSCs increase expression levels of EGFR and its heterodimerization partner HER2 during the course of osteogenic differentiation. tEGF substrata increased levels of phosphorylated EGFR and phosphorylated extracellular regulated kinase (ERK) compared to control substrata, and these elevations were associated with a twofold enhancement of MSC alkaline phosphatase activity at day 7 and matrix mineralization at day 21. Surprisingly, addition of soluble EGF (sEGF) to cells cultured on tEGF substrata reduces osteogenic differentiation, even though EGFR signaling is more strongly activated in acute, short-term manner by sEGF treatment than by tEGF treatment. A striking concomitant result of the sEGF effects is near-complete downregulation of EGFR and HER2, demonstrating that the tEGF/EGFR interaction is dynamically reversible even though temporally sustained. Taken together, our results show that enhanced MSC osteogenic differentiation corresponds to a sustained combination of receptor expression and ligand presentation, both of which are maintained by tEGF.

Mechanisms of field cancerization in the human stomach: the expansion and spread of mutated gastric stem cells.

BACKGROUND & AIMS: How mutations are established and spread through the human stomach is unclear because the clonal structure of gastric mucosal units is unknown. Here we investigate, using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion, the clonality of the gastric unit and show how mutations expand in normal mucosa and gastric mucosa showing intestinal metaplasia. This has important implications in gastric carcinogenesis. METHODS: Mutated units were identified by a histochemical method to detect activity of cytochrome c oxidase. Negative units were laser-capture microdissected, and mutations were identified by polymerase chain reaction sequencing. Differentiated epithelial cells were identified by immunohistochemistry for lineage markers. RESULTS: We show that mtDNA mutations establish themselves in stem cells within normal human gastric body units, and are passed on to all their differentiated progeny, thereby providing evidence for clonal conversion to a new stem cell-derived unit-monoclonal conversion, encompassing all gastric epithelial lineages. The presence of partially mutated units indicates that more than one stem cell is present in each unit. Mutated units can divide by fission to form patches, with each unit sharing an indentical, mutant mtDNA genotype. Furthermore, we show that intestinal metaplastic crypts are clonal, possess multiple stem cells, and that fission is a mechanism by which intestinal metaplasia spreads. CONCLUSIONS: These data show that human gastric body units are clonal, contain multiple multipotential stem cells, and provide definitive evidence for how mutations spread within the human stomach, and show how field cancerization develops.

Comparative methylation profiles and telomerase biology of mouse multipotent adult germline stem cells and embryonic stem cells.

Recently, several groups described the isolation of mouse spermatogonial stem cells (SSCs) and their potential to develop to embryonic stem cell (ESC)-like cells, so-called multipotent germline stem cells (mGSCs). We were the first to derive such mGSCs from SSCs isolated from adult mouse testis and, therefore, called these mGSCs multipotent adult germline stem cells (maGSCs). Here, we comparatively analyzed gene-specific and global DNA methylation profiles as well as the telomerase biology of several maGSC and male ESC lines. We show that undifferentiated maGSCs are very similar to undifferentiated male ESCs with regard to global DNA methylation, methylation of pluripotency marker gene loci, telomerase activity and telomere length. Imprinted gene methylation levels were generally lower in undifferentiated maGSCs than in undifferentiated male ESCs, but, compared with undifferentiated mGSCs derived by other groups, more similar to those of male ESCs. Differentiation of maGSCs increased the methylation of three of the four analyzed imprinted genes to almost somatic methylation patterns, but dramatically decreased global DNA methylation. Our findings further substantiate the pluripotency of maGSCs and their potential for regenerative medicine.

Sustained expression of GRAIL during hematopoiesis results in dysregulated differentiation.

BACKGROUND/AIMS: Despite a sophisticated understanding of the hematopoietic developmental program at the transcriptional level, our understanding of the role of E3 ubiquitin ligases remains underdeveloped. The E3 ubiquitin ligase, GRAIL (RNF128), is expressed in the bone marrow, but its role is as yet undefined. In this study, we evaluate the effect of GRAIL expression during hematopoietic differentiation in vitro and in vivo. METHODS: Retroviral transduction of hematopoietic multipotent progenitor cells was used for methylcellulose colony assays and bone marrow reconstitution. RESULTS: Enforced expression of GRAIL in colony assays demonstrated skewing of hematopoietic lineage development toward granulocytic/monocytic cells. Bone marrow reconstitution experiments with progenitor cells expressing biologically varied levels of GRAIL demonstrated diminished erythropoiesis and megakaryopoiesis if GRAIL (endogenous or forced) is maintained throughout hematopoiesis. CONCLUSION: These data highlight a role for GRAIL during hematopoiesis and emphasize the importance of studying posttranslational effects to complement our current understanding of the transcriptional regulation of hematopoiesis.

Quantifying Senescence-Associated Phenotypes in Primary Multipotent Mesenchymal Stromal Cell Cultures.

Cellular senescence is a tumor suppressor mechanism that removes potentially neoplastic cells from the proliferative pool. Senescent cells naturally accumulate with advancing age; however, excessive/aberrant accumulation of senescent cells can disrupt normal tissue function. Multipotent mesenchymal stromal cells (MSCs), which are actively evaluated as cell-based therapy, can undergo replicative senescence or stress-induced premature senescence. The molecular characterization of MSCs senescence can be useful not only for understanding the clinical correlations between MSCs biology and human age or age-related diseases but also for identifying competent MSCs for therapeutic applications. Because MSCs are involved in regulating the hematopoietic stem cell niche, and MSCs dysfunction has been implicated in age-related diseases, the identification and selective removal of senescent MSC may represent a potential therapeutic target. Cellular senescence is generally defined by senescence-associated (SA) permanent proliferation arrest (SAPA) accompanied by persistent DNA damage response (DDR) signaling emanating from persistent DNA lesions including damaged telomeres. Alongside SA cell cycle arrest and DDR signaling, a plethora of phenotypic hallmarks help define the overall senescent phenotype including a potent SA secretory phenotype (SASP) with many microenvironmental functions. Due to the complexity of the senescence phenotype, no single hallmark is alone capable of identifying senescent MSCs. This protocol highlights strategies to validate MSCs senescence through the measurements of several key SA hallmarks including lysosomal SA Beta-galactosidase activity (SA-ßgal), cell cycle arrest, persistent DDR signaling, and the inflammatory SASP.

The doublecortin-expressing population in the developing and adult brain contains multipotential precursors in addition to neuronal-lineage cells.

Doublecortin (DCX) has recently been promulgated as a selective marker of cells committed to the neuronal lineage in both the developing and the adult brain. To explore the potential of DCX-positive (DCX+) cells more stringently, these cells were isolated by flow cytometry from the brains of transgenic mice expressing green fluorescent protein under the control of the DCX promoter in embryonic, early postnatal, and adult animals. It was found that virtually all of the cells (99.9%) expressing high levels of DCX (DCX(high)) in the embryonic brain coexpressed the neuronal marker betaIII-tubulin and that this population contained no stem-like cells as demonstrated by lack of neurosphere formation in vitro. However, the DCX+ population from the early postnatal brain and the adult subventricular zone and hippocampus, which expressed low levels of DCX (DCX(low)), was enriched for neurosphere-forming cells, with only a small subpopulation of these cells coexpressing the neuronal markers betaIII-tubulin or microtubule-associated protein 2. Similarly, the DCX(low) population from embryonic day 14 (E14) brain contained neurosphere-forming cells. Only the postnatal cerebellum and adult olfactory bulb contained some DCX(high) cells, which were shown to be similar to the E14 DCX(high) cells in that they had no stem cell activity. Electrophysiological studies confirmed the heterogeneous nature of DCX+ cells, with some cells displaying characteristics of immature or mature neurons, whereas others showed no neuronal characteristics whatsoever. These results indicate that DCX(high) cells, regardless of location, are restricted to the neuronal lineage or are bone fide neurons, whereas some DCX(low) cells retain their multipotentiality.

Generation of multipotent cell lines from a distinct population of male germ line stem cells.

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

4-tert-octylphenol regulates the differentiation of C3H10T1/2 cells into osteoblast and adipocyte lineages.

The aim of this study was to investigate whether 4-tert-octylphenol (OP) affects the differentiation of multipotent C3H10T1/2 cells, a cell line established from mouse embryonic connective tissue, into osteoblast and adipocyte lineages. Confluent C3H10T1/2 cells were incubated for 7 days with (OP-treated cultures) or without (control cultures) 15 microg/ml of OP. The 7-day treatment of confluent cells with OP decreased alkaline phosphatase activity by 81%, inhibited the expression of transforming growth factor beta2, and inhibited the morphological changes in cells to an osteoblastic appearance. These results indicate that the 7-day treatment of confluent C3H10T1/2 cells with OP inhibited their differentiation into osteoblasts. Since this treatment strongly induced the expression of peroxisome proliferator-activated receptor r (PPARr) but did not stimulate triacylglycerol (TG) accumulation in cells, C3H10T1/2 cells in the control and OP-treated cultures were incubated for 2 days with a hormone mixture (insulin [INS], dexamethasone, and 1-methyl-3-isobutylxanthine) and incubated for an additional 5 days with INS alone. The TG and adiponectin contents of the OP-treated cultures were 4.2 and 4.1 times higher, respectively, than those of the control cultures. There were many more Oil Red O-staining cells in the OP-treated cultures than in the control cultures. The expression of PPARr in the OP-treated cultures was higher than that in the control cultures. These results indicate that the OP-treated cultures contained a larger number of adipocytes than the control cultures. In conclusion, treatment of C3H10T1/2 cells with OP inhibited osteoblast differentiation, causing a lineage shift toward adipocytes.

Spontaneous transformation of a clonal population of dermis-derived multipotent cells in culture.

It is reported that adult multipotent stem cells can undergo spontaneous transformation after long-term in vitro culture. Understanding the molecular mechanisms involved in this spontaneous transformation process can help in the design of future therapeutic applications. By far, the transformation process of adult multipotent stem cell is not well understood. In this study, a tumorigenic cell line nominated TDMC1 was established from a clonal population of rat dermis-derived multipotent cells (DMCs) following spontaneous transformation in culture. The transformed cells could produce tumors with characteristics of fibrous histocytoma when they are inoculated subcutaneously into nude mice. The molecular profiles of the nontransformed DMCs and transformed cells were analyzed by a deoxyribonucleic acid microarray. Our results showed that the overactivation of the K-ras/mitogen-activated protein kinase kinase signaling pathway played an important role in the transformation process. These data may be helpful to explain, at least in part, the possible mechanism for the malignant transformation of adult multipotent cells.

In vitro analysis of multipotent mesenchymal stromal cells as potential cellular therapeutics in neurometabolic diseases in pediatric patients.

Multipotent mesenchymal stromal cells (MSCs) play an important role in stromal support for hematopoietic stem cells, immune modulation, and tissue regeneration. We investigated their potential as cellular therapeutic tools in neurometabolic diseases as a growing number of affected children undergo to bone marrow transplantation. MSCs were isolated from bone marrow aspirates and expanded ex vivo under various culture conditions. MSCs under optimal good medical practice (GMP)-conform culture conditions showed the typical morphology, immunophenotype, and plasticity. Biochemically, the activities of beta-hexosaminidase A, total beta-hexosaminidase, arylsulfatase A (ASA), and beta-galactosidase measured in MSCs were comparable to those in fibroblasts of healthy donors. These four enzymes were interesting for their expression in MSCs, as each of them is defective, respectively, in well-known neurometabolic diseases. We found that MSCs released significant amounts of ASA into the media. In coculture experiments, fibroblasts from patients with metachromatic leukodystrophy, who are deficient for ASA, took up a substantial amount of ASA that was released into the media from MSCs. Mannose-6-phosphate (M6P) inhibited this uptake, which was in accordance with the M6P receptor-mediated uptake of lysosomal enzymes. Taken together, we show that MSCs produce appreciable amounts of lysosomal enzyme activities, making these cells first-choice candidates for providing metabolic correction when given to enzyme-deficient patients. With the example of ASA, it was also shown that an enzyme secreted from MSCs is taken up by enzyme-deficient patient fibroblasts. Given the plasticity of MSCs, these cells represent an interesting add-on option for cellular therapy in children undergoing bone marrow transplantation for lysosomal storage diseases and other neurometabolic diseases.

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents ⩽10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1-2% of total c-kitpos myocardial cells, when stimulated with TGF-ß/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.