Human Fetal Mesenchymal Stem Cells Research Articles

Migration of Human Fetal Bone Marrow-derived Mesenchymal Stem Cells: Possible Involvement of GPCR and MMPs

Previously, we have demonstrated that not all adult human bone marrow-derived mesenchymal stem cells can migrate efficiently towards tumor cells. In the current study, we attempt to address whether different samples of human fetal bone marrow-derived mesenchymal stem cells (hfMSC) also exhibit different migratory capacities toward tumors as was found to be the case with adult human MSC. Further, we are keen to explore the underlying mechanism of how hfMSC home to tumor cells. Using modified Boyden chamber assay, we demonstrated that hfMSC migrate at an efficiency comparable or better than the highly migratory adult MSC. Unlike the adult MSC, the extent of migration was not correlated to the expression and activity of MMP1. Rather, it appeared to be dependent in part on the PAR1 expression which may in turn be modulated by GPCR signal pathways. Additional evaluation will need to be done to further confirm the exact migratory mechanism. Nevertheless, hfMSC may potentially serve as equally efficient carriers of therapeutic genes to tumors.

The effects of culture on genomic imprinting profiles in human embryonic and fetal mesenchymal stem cells

Human embryonic stem (hES) cells and fetal mesenchymal stem cells (fMSC) offer great potential for regenerative therapy strategies. It is therefore important to characterise the properties of these cells in vitro. One major way the environment impacts on cellular physiology is through changes to epigenetic mechanisms. Genes subject to epigenetic regulation via genomic imprinting have been characterised extensively. The integrity of imprinted gene expression therefore provides a measurable index for epigenetic stability. Allelic expression of 26 imprinted genes and DNA methylation at associated differentially methylated regions (DMRs) was measured in fMSC and hES cell lines. Both cell types exhibited monoallelic expression of 13 imprinted genes, biallelic expression of six imprinted genes, and there were seven genes that differed in allelic expression between cell lines. fMSCs exhibited the differential DNA methylation patterns associated with imprinted expression. This was unexpected given that gene expression of several imprinted genes was biallelic. However, in hES cells, differential methylation was perturbed. These atypical methylation patterns did not correlate with allelic expression. Our results suggest that regardless of stem cell origin, in vitro culture affects the integrity of imprinted gene expression in human cells. We identify biallelic and variably expressed genes that may inform on overall epigenetic stability. As differential methylation did not correlate with imprinted expression changes we propose that other epigenetic effectors are adversely influenced by the in vitro environment. Since DMR integrity was maintained in fMSC but not hES cells, we postulate that specific hES cell derivation and culturing practices result in changes in methylation at DMRs.

A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering

Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4–2.5x, p < 0.05) in large 785 mm 3 macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2–1.6×, p < 0.01) and calcium deposition (1.3–2.3×, p < 0.01). We developed a Micro CT quantification method which demonstrated homogenous distribution of hfMSC in BXR bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds.

Isolation of cancer stem cells from transformed human mesenchymal stem cell line F6

Cancer stem cells (CSCs) have the characteristics of self-renewal, unlimited proliferation, and initiating new tumors. However, the origin of CSCs is still controversial. F6, a tumor cell line transformed from human fetal mesenchymal stem cells, was established in our previous study. Whether CSCs exist in this mutated cell line remains unclear. In the present study, we isolated CSCs from F6 cells based on CD133 expression using flow cytometry and investigated the biological characteristics of CD133+ F6 cells (F6-CD133+). We observed that the F6-CD133+ cells grew faster and had a higher capacity of colony formation than the F6-CD133- cells and parental F6 cells in vitro. In addition, F6-CD133+ cells had a higher tumorigenic ability than F6-CD133- cells in vivo since 1,000 F6-CD133+ cells were able to form tumors in nude mice. Cell viability assay revealed that F6-CD133+ cells were more sensitive to cisplatin while less to 5-fluorouracil. Furthermore, gene expression profile analysis showed that there were 673 differentially expressed genes between F6-CD133+ and F6-CD133- cells, many of which were involved in key cell signaling pathways. Taken together, our findings confirm that F6 cells contain a population of CSCs that contribute to its heterogeneity and tumorigenic potential, indicating that transformed stem cells could be the source of CSCs, and targeting this population may lead to more effective tumor treatments.

Identification of candidate surface antigens for non-invasive prenatal diagnosis by comparative global gene expression on human fetal mesenchymal stem cells

Transplacental passage of circulating first-trimester fetal mesenchymal stem cells (fMSC) raises the prospect of harvesting fetal cells in maternal blood. Despite high sensitivity in model systems, negative selection and culture strategies yield fMSC only rarely in post-termination maternal blood. The different adhesion molecule profile of fMSC to competitor maternal cell types suggests that improved positive selection strategies may facilitate non-invasive prenatal diagnosis. We aimed to identify surface antigens specific to fMSC and not maternal peripheral blood lymphocytes (PBL), using genome-wide analysis of actively expressed transcripts. Maternal PBL and fMSC cultured from first-trimester blood, liver and bone marrow were assessed for global gene expression by Affymetrix U133Plus2.0 arrays. Data were analysed using Affymetrix GCOS01.2. Transcripts present in all fMSC (n = 9) but absent in all PBL samples (n = 3) were selected for further analysis of cell-surface membrane molecules by RT-PCR and immunocytochemistry. Of 1544 genes expressed in fMSC and not maternal PBL, filtering for cell-surface molecules yielded 159 genes. Of these, 29 had a mean expression ratio of >300 (P < 0.001), which represented 18 unique genes, and their positive expression in all fMSC samples was confirmed by RT-PCR. Candidates for non-invasive prenatal diagnosis were chosen for further analysis by immunocytochemistry. Surface expression of OSMR and COL1 proteins on all fMSC, but no maternal PBL was confirmed. Identification of novel surface antigens on circulating human fMSC and not maternal PBL facilitates positive selection strategies for isolating fMSC for non-invasive prenatal diagnosis.

Human fetal mesenchymal stem cells differentiate into brown and white adipocytes: a role for ERRα in human UCP1 expression

We have investigated the ability of fetal mesenchymal stem cells (fMSCs) to differentiate into brown and white adipocytes and compared the expression of a number of marker genes and key regulatory factors. We have shown that the expression of key adipocyte regulators and markers during differentiation is similar to that in other human and murine adipocyte models, including induction of PPARγ2 and FABP4. Notably we found that the pre-adipocyte marker Pref-1, is induced early in differentiation and then declines markedly as the process continues, suggesting that fMSCs first acquire pre-adipocyte characteristics as they commit to the adipogenic lineage, prior to their differentiation into mature adipocytes. After adipogenic induction, some stem cell isolates differentiated into cells resembling brown adipocytes and others into white. Detailed investigation of one isolate showed that the novel brown fat determining factor PRDM16 is expressed both before and after differentiation. Importantly these cells exhibited elevated basal UCP-1 expression, which was dependent on the activity of the orphan nuclear receptor ERRα, highlighting a novel role for ERRα in human brown fat. Thus fetal MSCs represent a useful in vitro model for human adipogenesis, and provide opportunities to study the stages prior to commitment to the adipocyte lineage. They also offer invaluable insights into the characteristics of human brown fat.

Derivation and characterization of human fetal MSCs: An alternative cell source for large-scale production of cardioprotective microparticles

The therapeutic effects of mesenchymal stem cells (MSCs) transplantation are increasingly thought to be mediated by MSC secretion. We have previously demonstrated that human ESC-derived MSCs (hESC-MSCs) produce cardioprotective microparticles in pig model of myocardial ischemia/reperfusion (MI/R) injury. As the safety and availability of clinical grade human ESCs remain a concern, MSCs from fetal tissue sources were evaluated as alternatives. Here we derived five MSC cultures from limb, kidney and liver tissues of three first trimester aborted fetuses and like our previously described hESC-derived MSCs; they were highly expandable and had similar telomerase activities. Each line has the potential to generate at least 1016–19 cells or 107–10 doses of cardioprotective secretion for a pig model of MI/R injury. Unlike previously described fetal MSCs, they did not express pluripotency-associated markers such as Oct4, Nanog or Tra1-60. They displayed a typical MSC surface antigen profile and differentiated into adipocytes, osteocytes and chondrocytes in vitro. Global gene expression analysis by microarray and qRT-PCR revealed a typical MSC gene expression profile that was highly correlated among the five fetal MSC cultures and with that of hESC-MSCs (r2>0.90). Like hESC-MSCs, they produced secretion that was cardioprotective in a mouse model of MI/R injury. HPLC analysis of the secretion revealed the presence of a population of microparticles with a hydrodynamic radius of 50–65 nm. This purified population of microparticles was cardioprotective at ∼1/10 dosage of the crude secretion.

Effect of 5-azacytidine induction duration on differentiation of human first-trimester fetal mesenchymal stem cells towards cardiomyocyte-like cells☆

The aim of this study is to investigate effects of 5-azacytidine (5-aza) induction duration on differentiation of bone marrow mesenchymal stem cells (MSCs) from human first-trimester abortus (hfMSCs) towards cardiomyocyte-like cells. hfMSCs were stimulated with 10 micromol/l 5-aza for 24 h (group A), 48 h (group B) and 21 days (group C), respectively. During the induction, 30-40% of the cells gradually enlarged, elongated, connected with adjoining cells and formed myotube-like structures, branches and string-bead-like nuclei. Some of the cells congregated into cell clusters or strips. After the induction, numerous myofilaments in the cytoplasm and conjunction of intercalated disc-like structure between adjoining cells were observed. The induced cells expressed messenger ribonucleic acids (mRNAs) and proteins of myocardium-specific alpha-actin, sarcomeric beta-myocin heavy chain and troponin-T. The positive cell percentages for the three antigens in group C were each significantly higher than those antigens in group A and B (P<0.01) and the cell population doubling time (PDT) of group C was longer than those of group A and B (P<0.01). These indicate that 21-d induction with 10 micromol/l 5-aza slows down proliferation speed of hfMSCs but increases differentiation rate of hfMSCs into cardiomyocyte-like cells if compared with 24-48 h induction.

Neo-vascularization and bone formation mediated by fetal mesenchymal stem cell tissue-engineered bone grafts in critical-size femoral defects

Tissue-engineered bone grafts (TEBG) require highly osteogenic cell sources for use in fracture repair applications. Compared to other sources of mesenchymal stem cells (MSC), human fetal MSC (hfMSC) have recently been shown to be more proliferative and osteogenic. We studied the functional performance of hfMSC-mediated TEBG in 7 mm rat femoral critical-sized bone defects (CSD). Dynamically-cultured and osteogenically-primed hfMSC seeded onto macroporous poly-ɛ-caprolactone tri-calcium phosphate scaffolds were transplanted into CSDs. After 12 weeks, hfMSC-mediated TEBG induced 2.1× more new bone formation (43.3 ± 10.5 vs. 21.0 ± 7.4 mm 3, p < 0.05), with greater compact and woven bone, and a 9.8× increase in stiffness (3.9 ± 1.7 vs. 0.4 ± 0.3 mNm/degree, p < 0.05) compared to acellular scaffolds, such that only animals transplanted with TEBG underwent full fracture repair of the CSD. Although hfMSC survived for <4 weeks, by 4 weeks they were associated with a 3.9× larger vasculature network in the defect area (35.2 ± 11.1 vs. 6.5 ± 3.6 mm 3 p < 0.05), suggesting an important role for hfMSC in the promotion of neo-vasculogenesis. We speculate that hfMSC-mediated healing of the CSD by stimulating neo-vascularization through as yet undetermined mechanisms. This proof-of-principle study demonstrates the utility of primitive MSC for bone regeneration, and may be of relevance to vascularization in other areas of regenerative medicine.

Microgel Iron Oxide Nanoparticles for Tracking Human Fetal Mesenchymal Stem Cells Through Magnetic Resonance Imaging

Stem cell transplantation for regenerative medicine has made significant progress in various injury models, with the development of modalities to track stem cell fate and migration post-transplantation being currently pursued rigorously. Magnetic resonance imaging (MRI) allows serial high-resolution in vivo detection of transplanted stem cells labeled with iron oxide particles, but has been hampered by low labeling efficiencies. Here, we describe the use of microgel iron oxide (MGIO) particles of diameters spanning 100-750 nm for labeling human fetal mesenchymal stem cells (hfMSCs) for MRI tracking. We found that MGIO particle uptake by hfMSCs was size dependent, with 600-nm MGIO (M600) particles demonstrating three- to sixfold higher iron loading than the clinical particle ferucarbotran (33-263 versus 9.6-42.0 pg iron/hfMSC; p < .001). Cell labeling with either M600 particles or ferucarbotran did not affect either cellular proliferation or tri-lineage differentiation into osteoblasts, adipocytes, and chondrocytes, despite differences in gene expression on a genome-wide microarray analysis. Cell tracking in a rat photothrombotic stroke model using a clinical 1.5-T MRI scanner demonstrated the migration of labeled hfMSCs from the contralateral cortex to the stroke injury, with M600 particles achieving a five- to sevenfold higher sensitivity for MRI detection than ferucarbotran (p < .05). However, model-related cellular necrosis and acute inflammation limited the survival of hfMSCs beyond 5-12 days. The use of M600 particles allowed high detection sensitivity with low cellular toxicity to be achieved through a simple incubation protocol, and may thus be useful for cellular tracking using standard clinical MRI scanners.

Differentiation of human fetal mesenchymal stem cells into cells with an oligodendrocyte phenotype

The potential of mesenchymal stem cells (MSC) to differentiate into neural lineages has raised the possibility of autologous cell transplantation as a therapy for neurodegenerative diseases. We have identified a population of circulating human fetal mesenchymal stem cells (hfMSC) that are highly proliferative and can readily differentiate into mesodermal lineages such as bone, cartilage, fat and muscle. Here, we demonstrate for the first time that primary hfMSC can differentiate into cells with an oligodendrocyte phenotype both in vitro and in vivo. By exposing hfMSC to neuronal conditioned medium or by introducing the pro-oligodendrocyte gene, Olig-2, hfMSC adopted an oligodendrocyte-like morphology, expressed oligodendrocyte markers and appeared to mature appropriately in culture. Importantly we also demonstrate the differentiation of a clonal population of hfMSC into both mesodermal (bone) and ectodermal (oligodendrocyte) lineages. In the developing murine brain transplanted hfMSC integrated into the parenchyma but oligodendrocyte differentiation of these naïve hfMSC was very low. However, the proportion of cells expressing oligodendrocyte markers increased significantly (from 0.2% to 4%) by pre-exposing the cells to differentiation medium in vitro prior to transplantation. Importantly, the process of in vivo differentiation occurred without cell fusion. These findings suggest that hfMSC may provide a potential source of oligodendrocytes for study and potential therapy.

A biaxial rotating bioreactor for the culture of fetal mesenchymal stem cells for bone tissue engineering

The generation of effective tissue engineered bone grafts requires efficient exchange of nutrients and mechanical stimulus. Bioreactors provide a manner in which this can be achieved. We have recently developed a biaxial rotating bioreactor with efficient fluidics through in-silico modeling. Here we investigated its performance for generation of highly osteogenic bone graft using polycaprolactone–tricalcium phosphate (PCL–TCP) scaffolds seeded with human fetal mesenchymal stem cell (hfMSC). hfMSC scaffolds were cultured in either bioreactor or static cultures, with assessment of cellular viability, proliferation and osteogenic differentiation in vitro and also after transplantation into immunodeficient mice. Compared to static culture, bioreactor-cultured hfMSC scaffolds reached cellular confluence earlier (day 7 vs. day 28), with greater cellularity (2×, p < 0.01), and maintained high cellular viability in the core, which was 2000 μm from the surface. In addition, bioreactor culture was associated with greater osteogenic induction, ALP expression (1.5× p < 0.01), calcium deposition (5.5×, p < 0.001) and bony nodule formation on SEM, and in-vivo ectopic bone formation in immunodeficient mice (3.2×, p < 0.001) compared with static-cultured scaffolds. The use of biaxial bioreactor here allowed the maintenance of cellular viability beyond the limits of conventional diffusion, with increased proliferation and osteogenic differentiation both in vitro and in vivo, suggesting its utility for bone tissue engineering applications.

Isolation and culture of bone marrow mesenchymal stem cells from human fetus and their biological properties

AbstractThe population doubling number (70–80 times) of human fetal bone marrow mesenchymal stem cells (BMMSCs) is about two times higher than that (30–40 times) of adult BMMSCs, and their differentiation capacity is superior to that of their adult counterparts. In this study, BMMSCs were isolated from long bones of 2- to 3-month-old human abortuses by rinsing and selectively culturing whole marrow cells. Basic medium and serum concentration were optimized and growth curves plotted, both by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] reduction assay. Isolated cells were identified by flow cytometry and immunocytochemistry for their antigen markers. The biosafety of isolated cells was evaluated by karyotype analysis and a tumour-forming experiment. The results indicated that lengthwise scissoring of fetal long bones and rinsing of their marrow cells was practical and useful for recovery of BMMSCs from the investigated human abortuses. In this experiment, α-MEM (minimum essential medium, alpha medium)+20% FCS (fetal calf serum) was the best for in vitro culture of BMMSCs. The third-passage BMMSCs expressed Oct4, SSEA3 and SSEA4 besides the surface markers of their adult counterparts. The population doubling time of the BMMSCs of passage 6, 12 and 24 were 34, 36 and 40 h, respectively. Cells in all passages showed a diploid karyotype and formed no tumours in nude mice. The BMMSCs used in this study proved to be biologically safe and ideal seed cells for research on human tissue engineering and regeneration medicine.

First trimester embryo-fetoscopic and ultrasound-guided fetal blood sampling for ex vivo viral transduction of cultured human fetal mesenchymal stem cells

Intrauterine stem cell transplantation is a promising approach for early onset genetic diseases. However, its utility is limited by the development of the fetal immune system after 14 weeks gestation. An ex vivo gene therapy approach targeting autologous first trimester stem cells to replace the missing or defective gene product should overcome this barrier. We investigated the feasibility of harvesting circulating first trimester human fetal mesenchymal stem cells (hfMSCs) for ex vivo gene therapy. Thin-gauge embryofetoscopic-directed or ultrasound-guided blood sampling (FBS) was performed in 18 pre-termination fetuses at a mean of 10(+0) (range 7(+2) to 13(+4)) weeks gestation through extra-fetal vessels. Harvested blood was plated for isolation of hfMSC and transduced by lentiviruses. FBS was successful in 12/18 procedures (67%). Success rates were comparable in fetoscopic (4/6) and ultrasound-guided (8/12) procedures, but procedural time was shorter in the ultrasound-guided arm (P = 0.01). Fetal bradycardia occurred post-FBS in 33% and 25% of fetoscopic and ultrasound cases, respectively, 5 min post-procedure. hfMSCs were isolated in two-thirds of cases, with high efficiency lentiviral transduction achieved without affecting short-term cell renewal. This phase-one study demonstrates the feasibility of the ex vivo fetal gene therapy approach, in which harvested hfMSCs are genetically manipulated prior to infusion back into the fetus where they should engraft and home to injured tissues. The fetal ex vivo gene therapy paradigm is also of relevance to haemopoietic stem cells to treat inherited haematological diseases. Optimization of stem cell harvest and longer-term safety is required before translation into clinical trials in ongoing pregnancies.

In Vitro Hepatic Differentiation of Mesenchymal Stem Cells from Human Fetal Bone Marrow

We examined whether human fetal mesenchymal stem cells (FMSCs) derived from fetal bone marrow were able to differentiate into functional hepatocyte-like cells in vitro. The surface phenotype of FMSCs was characterized by flow cytometry. To induce hepatic differentiation of FMSCs, we added hepatocyte growth factor, basic fibroblast growth factor and oncostatin M into the cell culture medium. After 21 days of hepatocyte induction, FMSCs expressed the hepatocyte-specific markers, alpha-fetoprotein and cytokeratin 18, as demonstrated by immunofluorescence staining. Differentiated FMSCs also demonstrated in vitro functions characteristic of liver cells, including albumin production, urea secretion and glycogen storage. In conclusion, fetal bone marrow-derived FMSCs are able to differentiate into functional hepatocytelike cells and may serve as a source of cells for liver disease therapy.

Intrauterine transplantation of human fetal mesenchymal stem cells from first-trimester blood repairs bone and reduces fractures in osteogenesis imperfecta mice

AbstractThe inherited skeletal dysplasia osteogenesis imperfecta (OI) results in multiple fractures and is currently treated empirically. We transplanted human first-trimester fetal blood mesenchymal stem cells (MSCs) into homozygous oim mice in utero. This resulted in a two-thirds reduction in long bone fractures (P < .01), with fewer fractures per mouse (median 1, range 0-2 in mice that received transplants vs median 3, range 1-5 in mice that did not receive transplants by 12 weeks, P < .01). Nearly all mice that did not receive transplants had fractures (47 [97.9%] of 48), in contrast to 17 (58.6%) of 29 4- to 12-week-old mice that received transplants (P < .01). Transplantation was associated with increased bone strength (P < .01), thickness (P < .01), and length (P < .01), and normalization/reduction of growth plate height in 4- to 12-week-old oim was reduced in mice that underwent transplantion (P < .001). More donor cells were found in bone tissues compared with other organs (P < .001), with cells clustered in areas of active bone formation and remodeling, and at sites of fracture healing. Donor cells found in the bone expressed osteoblast lineage genes, and produced the extracellular bone structural protein osteopontin. Finally, MSC transplantation decreased bone hydroxyproline content. In conclusion, intrauterine transplantation of fetal MSCs markedly reduced fracture rates and skeletal abnormalities in a mouse model of the intermediate severity type III OI, suggesting a scientific basis for MSC treatment of affected human fetuses.

Osterix Induces Osteogenic Gene Expression but not Differentiation in Primary Human Fetal Mesenchymal Stem Cells

The transcription factor osterix (Osx) is a key regulator of osteoblast differentiation and induces bone formation in embryonic but not adult stem cells. We investigated the effect of up-regulating Osx on an intermediate stem cell type, first trimester fetal mesenchymal stem cells (MSCs), which are more expandable than adult MSCs. Human fetal (hf ) MSCs were transduced with a lentiviral vector encoding human Osx. In undifferentiating MSCs cultures, forced expression of Osx stimulated osteopontin and alkaline phosphatase expression. However, Osx did not up-regulate osteocalcin, a late marker of osteoblast differentiation or result in extracellular calcium crystals, indicating that Osx does not directly mediate terminal differentiation in primary hfMSCs. To understand the downstream effects of Osx expression in primary hfMSCs, we next investigated the regulatory relationship between Osx, and the transcription factors Dlx5, Runx2, and Msx2. Osx induced Dlx5 but did not affect Runx2 and Msx2, whereas stealth ribonucleic acid interference of Osx inhibited Dlx5 without affecting expression of Runx2 and Msx2. In conclusion, Osx regulates osteogenic gene expression in hfMSCs but is insufficient to induce terminal osteogenic differentiation.

Adipocyte differentiation induced using nonspecific siRNA controls in cultured human mesenchymal stem cells

RNA interference (RNAi) is gene silencing induced by double-stranded RNA of 21-23 nucleotides in length, termed small interfering RNA, or siRNA. RNAi-based techniques have been widely applied to elucidate gene function, identify drug targets, and used in trials as a promising adjunct to silence disease-causing genes. However, emerging evidence suggests unexpected changes in expression of untargeted genes as a consequence of an off-target effect by RNAi in mammalian cells. To date, our understanding of such effects on stem cells is limited. We transfected human fetal femur-derived mesenchymal stem cells using commercially available nonspecific siRNA controls and examined adipocyte differentiation in the cells using morphology, histochemistry, and quantitative real-time PCR to examine the expression of key genes for adipogenic or osteogenic differentiation. We report here the induction of adipocyte differentiation in human mesenchymal stem cells using nonspecific siRNAs raising concerns as to the specificity of RNAi in stem cells and, critically, a need to understand and delineate the rules governing the specificity of RNAi.

Experimental intravenous cell therapy of acute and chronic renal failure

The therapeutic effect of intravenous injection of human fetal bone marrow mesenchymal stem cells or summary culture of kidney cells were studied on models of chronic or acute renal failure in outbred albino rats. Both cell types promoted improvement and normalization of the renal function in rats with stable chronic renal insufficiency (2 weeks after kidney cell injection, 1 month after bone marrow mesenchymal stem cell injection). Renal function remained normal or subnormal during the delayed period (3-3.5 months after injection). In rats with latent stage of chronic renal insufficiency, exacerbation was induced by additional 40-min ischemia. All rats receiving intravenous injection of saline died. Improvement of the functional parameters started 2 weeks after injection of kidney cells or bone marrow mesenchymal stem cells, and normalization was observed after 1.1-5 months. During the delayed period (after 3-4 months), functional parameters retained at normal or subnormal levels. In experimental series III, all rats with acute renal failure intravenously injected with saline (control) died from uremia on days 2-4. After injection of kidney cells 50% rats survived and renal function in these animals returned to normal after 2 weeks. After injection of bone marrow mesenchymal stem cells 83% rats survived, functional parameters returned to normal after 3 weeks.

Widespread Distribution and Muscle Differentiation of Human Fetal Mesenchymal Stem Cells After Intrauterine Transplantation in Dystrophic mdx Mouse

Duchenne muscular dystrophy (DMD) is a common X-linked disease resulting from the absence of dystrophin in muscle. Affected boys suffer from incurable progressive muscle weakness, leading to premature death. Stem cell transplantation may be curative, but is hampered by the need for systemic delivery and immune rejection. To address these barriers to stem cell therapy in DMD, we investigated a fetal-to-fetal transplantation strategy. We investigated intramuscular, intravascular, and intraperitoneal delivery of human fetal mesenchymal stem cells (hfMSCs) into embryonic day (E) 14-16 MF1 mice to determine the most appropriate route for systemic delivery. Intramuscular injections resulted in local engraftment, whereas both intraperitoneal and intravascular delivery led to systemic spread. However, intravascular delivery led to unexpected demise of transplanted mice. Transplantation of hfMSCs into E14-16 mdx mice resulted in widespread long-term engraftment (19 weeks) in multiple organs, with a predilection for muscle compared with nonmuscle tissues (0.71% vs. 0.15%, p < .01), and evidence of myogenic differentiation of hfMSCs in skeletal and myocardial muscle. This is the first report of intrauterine transplantation of ontologically relevant hfMSCs into fully immunocompetent dystrophic fetal mice, with systemic spread across endothelial barriers leading to widespread long-term engraftment in multiple organ compartments. Although the low-level of chimerism achieved is not curative for DMD, this approach may be useful in other severe mesenchymal or enzyme deficiency syndromes, where low-level protein expression may ameliorate disease pathology.