Trafficking Of Mesenchymal Stem Cells Research Articles

Involvement of various chemokine/chemokine receptor axes in trafficking and oriented locomotion of mesenchymal stem cells in multiple sclerosis patients.

Multiple sclerosis (MS) is a specific type of chronic immune-mediated disease in which the immune responses are almost run against the central nervous system (CNS). Despite intensive research, a known treatment for MS disease yet to be introduced. Thus, the development of novel and safe medications needs to be considered for the disease management. Application of mesenchymal stem cells (MSCs) as an emerging approach was recruited forthe treatment of MS. MSCs have several sources and they can be derived from the umbilical cord, adipose tissue, and bone marrow. Chemokines are low molecular weight proteins that their functional activities are achieved by binding to the cell surface G protein-coupled receptors (GPCRs). Chemokine and chemokine receptors are of the most important and effective molecules in MSC trafficking within the different tissues in hemostatic and non-hemostatic circumstances. Chemokine/chemokine receptor axes play a pivotal role in the recruitment and oriented trafficking of immune cells both towards and within the CNS and it appears that chemokine/chemokine receptor signaling may be the most important leading mechanisms in the pathogenesis of MS. In this article, we hypothesized that the chemokine/chemokine receptor axes network have crucial and efficacious impacts on behavior of the MSCs, nonetheless, the exact responsibility of these axes on the targeted tropism of MSCs to the CNS of MS patients yet remained to be fully elucidated. Therefore, we reviewed the ability of MSCs to migrate and home into the CNS of MS patients via expression of various chemokine receptors in response to chemokines expressed by cells of CNS tissue, to provide a great source of knowledge.

Inflammatory Mediators in Glioma Microenvironment Play a Dual Role in Gliomagenesis and Mesenchymal Stem Cell Homing: Implication for Cellular Therapy.

Glioblastoma is the most aggressive malignant primary brain tumor, with a dismal prognosis and a devastating overall survival. Despite aggressive surgical resection and adjuvant treatment, average survival remains approximately 14.6 months. The brain tumor microenvironment is heterogeneous, comprising multiple populations of tumor, stromal, and immune cells. Tumor cells evade the immune system by suppressing several immune functions to enable survival. Gliomas release immunosuppressive and tumor-supportive soluble factors into the microenvironment, leading to accelerated cancer proliferation, invasion, and immune escape. Mesenchymal stem cells (MSCs) isolated from bone marrow, adipose tissue, or umbilical cord are a promising tool for cell-based therapies. One crucial mechanism mediating the therapeutic outcomes often seen in MSC application is their tropism to sites of injury. Furthermore, MSCs interact with host immune cells to regulate the inflammatory response, and data points to the possibility of using MSCs to achieve immunomodulation in solid tumors. Interleukin 1β, interleukin 6, tumor necrosis factor α, transforming growth factor β, and stromal cell–derived factor 1 are notably up-regulated in glioblastoma and dually promote immune and MSC trafficking. Mesenchymal stem cells have widely been regarded as hypoimmunogenic, enabling this cell-based administration across major histocompatibility barriers. In this review, we will highlight (1) the bidirectional communication of glioma cells and tumor-associated immune cells, (2) the inflammatory mediators enabling leukocytes and transplantable MSC migration, and (3) review preclinical and human clinical trials using MSCs as delivery vehicles. Mesenchymal stem cells possess innate abilities to migrate great distances, cross the blood-brain barrier, and communicate with surrounding cells, all of which make them desirable “Trojan horses” for brain cancer therapy.

Delayed release of chemokine CCL25 with bioresorbable microparticles for mobilization of human mesenchymal stem cells

Chemokines are guiding cues for directional trafficking of mesenchymal stem cells (MSC) upon injury and local chemokine delivery at injury sites is an up-to-date strategy to potentiate and prolong recruitment of MSC.In this study we present the chemokine CCL25, also referred to as thymus-expressed chemokine, to mobilize human MSC along positive but not along negative gradients. We hence proceeded to design a biodegradable and injectable release device for CCL25 on the basis of poly(lactic-co-glycolic acid) (PLGA). The conducted studies had the objective to optimize PLGA microparticle fabrication by varying selected formulation parameters, such as polymer type, microparticle size and interior phase composition. We found that microparticles of DV,50∼75 µm and fabricated using end-capped polymers, BSA as carrier protein and vortex mixing to produce the primary emulsion yielded high chemokine loading and delayed CCL25 release. To determine bioactivity, we investigated CCL25 released during the microparticle erosion phase and showed that deacidification of the release medium was required to induce significant MSC mobilization.The designed PLGA microparticles represent an effective and convenient off-the-shelf delivery tool for the delayed release of CCL25. However, continuative in vivo proof-of-concept studies are required to demonstrate enhanced recruitment of MSC and/or therapeutical effects in response to CCL25 release microparticles. Statement of SignificanceWith the discovery of chemokines, particularly CXCL12, as stimulators of stem cell migration, the development of devices that release CXCL12 has proceeded quickly in the last few years. In this manuscript we introduce CCL25 as chemokine to induce mobilization of human MSC. This study proceeds to demonstrate how selection of key formulation parameters of CCL25 loading into PLGA microparticles exerts considerable influence on CCL25 release. This is important for a broad range of efforts in in situ tissue engineering where the candidate chemokine and the delivery device need to be selected carefully. The use of such a cell-free CCL25 release device may provide a new therapeutic option in regenerative medicine.

Mesenchymal stem cell biodistribution, migration, and homing in vivo.

Mesenchymal stem cells (MSCs) were originally defined by their capacity to differentiate into various connective tissue lineages as well as support hematopoiesis in vitro via the production of various cytokines, chemokines, and adhesion molecules [1, 2]. During the past decade, MSCs have been shown to exhibit angiogenic, trophic, anti-inflammatory, and immunomodulatory activity using a variety of experimental paradigms [3–6]. Together with their easy availability and amenability to large-scale expansion ex vivo, these properties have propelled MSC-based therapies to the forefront of regenerative medicine and immune regulatory cell therapy. Currently, MSCs from a variety of tissue sources are being evaluated in over 200 clinical trials for the treatment of a diverse array of disease indications. Completed Phase I and II clinical trials have reported statistically significant benefits in patients with steroid-resistant graft versus host disease [7], severe systemic lupus erythematosus [8], complex perianal fistulas [9], and ischemic cardiomyopathy [10]. However, not all trials have met their primary endpoint of efficacy and while many factors contribute to suboptimal patient outcomes, key among them are the molecular mechanisms that govern MSC engraftment, homing, and biodistribution in vivo. Indeed, despite rapid progress in describing the therapeutic potency of MSCs in experimental animal models of disease, progress in understanding their biodistribution and mechanism of action in vivo has been slow to develop. For example, robust methodologies to track the fate of MSCs in vivo are critical toward establishing their tissue tropism, duration of engraftment, and rates of clearance. In addition, the identification of endogenous factors that function as chemoattractants and repellents also plays critical roles in directing transplanted cells to sites of tissue injury. Moreover, a clearer understanding of the signaling axes that regulate MSC trafficking in vivo would provide a means to direct cells to specific tissue and organs, thereby increasing overall efficacy of MSC-based therapies. The latter may also provide a means to mobilize endogenous MSCs and enhance their regenerative and immune regulatory properties. Finally, cellular crosstalk and cell-to-cell interactions also likely affect the biodistribution and survival of exogenously administered MSCs, but scant information exists regarding these processes in vivo. In fact, it is a subject of debate whether MSCs localize to tissue due to passive entrapment in small vessels, particularly in lung, or if cells employ active mechanisms similar to leukocytes to home to specific tissues. Therefore, continued study into the mechanism that regulates trafficking of endogenous and transplanted MSCs will shed novel insight into basic MSC biology and lead to the development of more potent cell-based therapies.

SP0167 Bone Marrow and Synovial Fluid Stem Cells: In Vivo as Drivers of Spontaneous Tissue Repair

Bone marrow and synovial fluid stem cells in vivo as drivers of spontaneous tissue repairRegenerative Medicine approaches have entered multiple fields of Medicine including Orthopedics, Neurodegenerative Disease, Liver disease and...

Effect of a CCR1 receptor antagonist on systemic trafficking of MSCs and polyethylene particle-associated bone loss

Particle-associated periprosthetic osteolysis remains a major issue in joint replacement. Ongoing bone loss resulting from wear particle-induced inflammation is accompanied by continued attempts at bone repair. Previously we showed that mesenchymal stem cells (MSCs) are recruited systemically to bone exposed to continuous infusion of ultra high molecular weight polyethylene (UHMWPE) particles. The chemokine–receptor axis that mediates this process is unknown. We tested two hypotheses: (1) the CCR1 receptor mediates the systemic recruitment of MSCs to UHMWPE particles and (2) recruited MSCs are able to differentiate into functional mature osteoblasts and decrease particle-associated bone loss. Nude mice were allocated randomly to four groups. UHMWPE particles were continuously infused into the femoral shaft using a micro-pump. Genetically modified murine wild type reporter MSCs were injected systemically via the left ventricle. Non-invasive imaging was used to assay MSC migration and bone mineral density. Bioluminescence and immunohistochemistry confirmed the chemotaxis of reporter cells and their differentiation into mature osteoblasts in the presence of infused particles. Injection of a CCR1 antagonist decreased reporter cell recruitment to the UHMWPE particle infusion site and increased osteolysis. CCR1 appears to be a critical receptor for chemotaxis of MSCs in the presence of UHMWPE particles. Interference with CCR1 exacerbates particle-induced bone loss.

Modulation of Stromal Cell-Derived Factor-1/CXC Chemokine Receptor 4 Axis Enhances rhBMP-2-Induced Ectopic Bone Formation

Enhancement of in vivo mobilization and homing of endogenous mesenchymal stem cells (MSCs) to an injury site is an innovative strategy for improvement of bone tissue engineering and repair. The present study was designed to determine whether mobilization by AMD3100 and/or local homing by delivery of stromal cell-derived factor-1 (SDF-1) enhances recombinant human bone morphogenetic protein-2 (rhBMP-2) induced ectopic bone formation in an established rat model. Rats received an injection of either saline or AMD3100 treatment 1 h before harvesting of bone marrow for in vitro colony-forming unit-fibroblasts (CFU-F) culture or the in vivo subcutaneous implantation of absorbable collagen sponges (ACSs) loaded with saline, recombinant human bone morphogenetic protein-2 (rhBMP-2), SDF-1, or the combination of SDF-1 and rhBMP-2. AMD3100 treatment resulted in a significant decrease in CFU-F number, compared with saline, which confirmed that a single systemic AMD3100 treatment rapidly mobilized MSCs from the bone marrow. At 28 and 56 days, bone formation in the explanted ACS was assessed by microcomputed tomography (μCT) and histology. At 28 days, AMD3100 and/or SDF-1 had no statistically significant effect on bone volume (BV) or bone mineral content (BMC), but histology revealed more active bone formation with treatment of AMD3100, loading of SDF-1, or the combination of both AMD3100 and SDF-1, compared with saline-treated rhBMP-2 loaded ACS. At 56 days, the addition of AMD3100 treatment, loading of SDF-1, or the combination of both resulted in a statistically significant stimulatory effect on BV and BMC, compared with the saline-treated rhBMP-2 loaded ACS. Histology of the 56-day ACS were consistent with the μCT analysis, exhibiting more mature and mineralized bone formation with AMD3100 treatment, SDF-1 loading, or the combination of both, compared with the saline-treated rhBMP-2 loaded ACS. The present study is the first that provides evidence of the efficacy of AMD3100 and SDF-1 treatment to stimulate trafficking of MSCs to an ectopic implant site, in order to ultimately enhance rhBMP-2 induced long-term bone formation.

Damage‐associated molecular patterns (DAMPs) and mesenchymal stem cells: A matter of attraction and excitement

Necrotic cell death is a typical feature of solid tumors leading to the release of necrotic products, also known as damage-associated molecular patterns (DAMPs), that enhance angiogenesis and prime the immune response. Among the DAMPs, particular attention has been focused on the DNA-binding molecule high-mobility group box 1 (HMGB-1) that can act as a chemoattractant and activator of granulocytes. Here, we discuss an article in this issue of the European Journal of Immunology that demonstrates that DAMPs promote both proliferation and trafficking of mesenchymal stem cells (MSCs), identifying HMGB-1 as a key factor in the regulation of these processes. Moreover, the study shows that DAMPs interfere with the expression of the immunosuppressive molecule indoleamine-2,3-dioxygenase in MSCs, and that the biological activity of HMGB-1 toward MSCs is abolished when HMGB-1 is oxidized. Based on the data from this, and other studies, we depict a model in which DAMPs released from necrotic tumor cells attract and stimulate local proliferation of MSCs that differentiate into tumor-associated fibroblasts promoting tumor growth and angiogenesis. Importantly, the hypoxic conditions of the tumor microenvironment may protect DAMPs from oxidation and thereby preserve their functionality.

Systemically Transplanted Human Bone Marrow Mesenchymal Stem Cells Primarily Traffic to Mesenteric Lymph Nodes

AbstractAbstract 2580Intravenously administered mesenchymal stem cells (MSCs) are trapped in pulmonary vascular bed and only few MSCs home to bone or other tissues in physiological or pathological conditions. Following intracardiac injection MSCs pass the lung barrier but their homing to bone and tissue localization is uncertain. The aim of the study was to investigate trafficking and exact localization of human MSCs following intracardiac injection into unchallenged mice and a xenograft bone tumor model. MSCs were isolated from human fetal bones (ABR Inc, Alameda CA) and expanded in DMEM-LG medium supplemented with 10% FBS. Global gene expression profiling revealed that the cultured MSCs were devoid of hematopoietic cells and expressed typical mesenchymal markers such as CD166, CD146 and CD90. We have previously shown that these MSCs are capable of differentiation into osteoblasts and adipocytes and retain their differentiation potential after multiple passages (Haematologica 2006). The MSCs were transduced with a luciferase/GFP reporter in a lentiviral vector and were maximally passaged 8 times before used in vivo. Detection of MSCs in mice was determined by live-animal imaging and ex vivo bioluminescence activity using the IVIS system, by microscopic examination of GFP-expressing cells and by immunohistochemistry for GFP. MSCs (1×106 cells/mouse) were intracardiacly injected into unconditioned SCID mice (n=8) using Dovetail Slide Micromanipulator that ensures accurate injection. Following 2 or 7 days after MSC injection to SCID mice, live-animal imaging revealed bioluminescence activity mainly in the mice abdomen but not bone, while ex vivo examination detected MSCs in various abdominal organs, primarily in reproductive organs, intestine and pancreas. Careful microscopic examination revealed localization of MSCs in draining lymph nodes attached to these organs by connective tissue. Immunohistochemistry showed GFP-expressing MSCs in the adjacent mesenteric lymph nodes but not within the organs. To confirm our findings, MSCs were intracardially injected into C57BL6 mice (n=6) that harbor functional lymph nodes. Evans blue dye which is known to accumulate in and identify lymph nodes, was injected into the rear footpad or lateral tail base of the mice, 3 hours after MSC injection and 30 minutes prior to bioluminescence and florescence analyses. The Evans blue dye and GFP positivity were co-localized, indicating specific trafficking of MSCs to lymph nodes. Culturing of the dissected lymph nodes resulted in release of GFP-expressing MSCs which regained their in vitro morphology. For testing MSCs trafficking in a xenograft model, we used our SCID-rab system constructed by implanting a 4-weeks old rabbit bone into which human myeloma cells were directly injected (Leukemia 2004; Blood 2007). In this model myeloma cells grow restrictively in the implanted bone. MSCs injected intracardiacly into SCID-rab mice were mostly found in mesenteric lymph nodes but were also detected in the myelomatous bone 72 hours after MSCs injection, validating the ability of tumor cells to attract MSCs and that these MSCs are capable of transmigration. We conclude that MSCs primarily traffic to draining lymph nodes, partially explaining their in vivo immunomodulatory activity, and that understanding the mechanism by which MSCs traffic to lymph nodes may help develop approaches to shift their homing to desired organs. Disclosures:No relevant conflicts of interest to declare.

Migration potential and gene expression profile of human mesenchymal stem cells induced by CCL25

Recruitment of mesenchymal stem cells (MSC) to tissue damages is a promising approach for in situ tissue regeneration. The physiological mechanisms and regulatory processes of MSC trafficking to injured tissue remain poorly understood. However, the pivotal role of chemokines in MSC recruitment has already been shown. The aim of this study was to determine the migratory potential and the gene expression profile of MSC stimulated with the CC chemokine CCL25 (TECK). Bone marrow derived human MSC were exposed to different doses of CCL25 in a standardized chemotaxis assay. Microarray gene expression profiling and pathway analysis were performed for CCL25 stimulated MSC. Maximum migration of MSC towards CCL25 was observed at 10 3 nM. Microarray analysis revealed an induction of molecules directly involved in chemotaxis and homing of bone marrow cells (CXCL1–3, CXCL8, PDE4B), cytoskeletal and membrane reorganisation (CXCL8, PLD1, IGFBP1), cellular polarity (PLD1), and cell movement (CXCL1–3, CXCL6, CXCL8, PTGS2, PDE4B, TGM2). Respective chemokine secretion was confirmed by protein membrane-array analysis. The activation of CXCR2 ligands (CXCL1–3, CXCL5–6, CXCL8) and a LIF-receptor/gp130 ligand (LIF) indicated an involvement of the respective signaling pathways during initiation of chemotaxis and migration. These results suggest CCL25 as a new potential candidate for further in situ regeneration approaches.

Ex Vivo Glycan Engineering of Membrane CD44 To Create HCELL Programs Human Mesenchymal Stem Cell Trafficking to Bone.

The proximate obstacle to realizing the potential benefits of stem cell-based regenerative therapeutics is to safely and efficiently deliver the cells where they are needed/required. Though transplantation of culture-expanded human mesenchymal stem cells (MSC) holds the potential for cure of generalized skeletal diseases such as osteogenesis imperfecta, clinical effectiveness has been constrained to date by the poor osteotropism (migration to bone) of infused MSCs. Tissue-specific migration is initiated by adhesive interactions mediated by molecules on the surface of blood-borne cells that are specialized to resist the shear forces of blood flow, the “homing receptors”. The recruitment of circulating cells to bone occurs within the marrow, at specialized marrow vessels that constitutively express E-selectin, a lectin that recognizes sialofucosylated determinants on its respective ligand(s). To gain insight on the expression and function of homing receptors on human MSC, we performed flow cytometry and Western blot analysis, together with parallel plate flow chamber studies under physiologic shear conditions. Whereas human hematopoietic progenitor cells (HPC) characteristically display the E-selectin ligands CLA and HCELL, which are α(2,3)-sialyl-α(1,3)-fucosylated glycoforms of PSGL-1 and CD44, respectively, biochemical and functional studies showed that human MSC express no E-selectin ligands and are devoid of PSGL-1. We also found that human MSC did not express many effectors of cellular trafficking to marrow present on HPC, such as LFA-1, LPAM-1 (α4β7), nor the chemokine receptor CXCR4, but did express VLA-4 and, notably, display a CD44 glycoform bearing α(2,3)-sialyllactosamine modifications that can serve as an acceptor for exogenous fucosylation. Employing an α(1,3)-fucosyltransferase preparation and enzymatic conditions specifically designed for treating live cells, the native human MSC surface CD44 glycoform was converted to the E-selectin ligand HCELL with commensurate induction of potent E-selectin-dependent shear-resistant rolling interactions on activated endothelium, without effects on MSC viability or multipotency. As observed by real-time intravital microscopy in immunocompromised (NOD/SCID) mouse hosts, intravenously infused HCELL+ human MSCs homed robustly to bone with marrow infiltrates evident within hours of infusion, whereas unmodified MSC and HCELL+ MSC treated with sialidase (which eliminates E-selectin binding) showed negligible rolling on marrow vessels and did not extravasate. Alignment of extravasated HCELL+ human MSC along the endosteal surface was observed, with rare differentiation to osteoblasts as evidenced by immunohistochemical staining for human osteocalcin. These results indicate that despite absence of CXCR4 and many other effectors of cellular trafficking to marrow, osteotropism of systemically administered human MSC was conferred by ex vivo cell surface carbohydrate modification of a single glycoprotein, CD44, rendering the potent E-selectin ligand HCELL. Our findings unveil a readily translatable roadmap for programming MSC trafficking, thus overcoming a critical barrier in use of these cells for regenerative therapeutics of systemic skeletal diseases.

Cellular Iron Metabolism Studies Demonstate Safety of Magnetic Tracking of Mesenchymal Stem Cells.

Background: Ferumoxides-protamine sulfate (Fe-Pro) complexes are used for intracellular magnetic labeling of mesenchymal stem cells (MSCs) to non invasively monitor cell trafficking and tissue distribution by in vivo magnetic resonance imaging (MRI). It has been previously shown that there was no short or long- term toxic effects on cell's viability, proliferation, reactive oxygen species and biological functions including differentiation capacity of MSCs, however the effects of dextran coated superparamagnetic iron oxide (SPIO) nanoparticles labeling on cellular iron metabolism and storage is unknown.Objective: Evaluate the effect of using Fe-Pro complexes for magnetic cell labeling in MSCs on the expression of transferrin receptor (TfR-1) and ferritin, proteins involved in intracellular iron metabolism and storage.Methods: Confluent MSCs were labeled with Fe-Pro complexes at the ratio of 100 ug/mL to 6 ug/mL. After labeling, cells were washed and cultured for 2 months. TfR-1 and ferritin expression were evaluated at the gene and protein levels in total cell lysates, on day 1, 3, 7, 14, 28 and 35 days post-labeling, by real-time polymerase chain reaction (RT-PCR) and Western blotting. MSCs were evaluated for the presence of iron by Prussian blue stain and light microscopy. In addition, TfR-1 and ferritin levels were determined for Fe-Pro labeled and unlabeled MSCs, allowed to slowly multiply, as a result of dividing the cells in half when they reached confluence.Results: All cells were Prussian blue positive on light microscopy following labeling with Fe-Pro. Labeling of MSCs grown to confluence resulted in short-term increase of TfR-1 mRNA (day 1 and day 3, p= 0.0056) without changes in TfR-1 protein levels. Fe- Pro labeled MSC demonstrated an increase in ferritin gene expression on day 7 and 14 (p=0.0003) compared to unlabeled cells, while protein levels were higher compared to unlabeled MSC at each time point (p=0.005). However, ferritin protein levels from Fe-Pro labeled MSCs were not significantly increased after 7 passages.Conclusions: Magnetic labeling of cells with FDA approved ferumoxides complexed to protamine sulfate elicited short-term changes in cellular iron metabolism and storage that was dependent on ability to divide. Fe-Pro labeling did not have any long-term implications validating the safety of Fe-Pro labeling technique for MSCs trafficking studies by MRI.

Dynamic Imaging of Allogeneic Mesenchymal Stem Cells Trafficking to Myocardial Infarction

Recent results from animal studies suggest that stem cells may be able to home to sites of myocardial injury to assist in tissue regeneration. However, the histological interpretation of postmortem tissue, on which many of these studies are based, has recently been widely debated. With the use of the high sensitivity of a combined single-photon emission CT (SPECT)/CT scanner, the in vivo trafficking of allogeneic mesenchymal stem cells (MSCs) colabeled with a radiotracer and MR contrast agent to acute myocardial infarction was dynamically determined. Redistribution of the labeled MSCs after intravenous injection from initial localization in the lungs to nontarget organs such as the liver, kidney, and spleen was observed within 24 to 48 hours after injection. Focal and diffuse uptake of MSCs in the infarcted myocardium was already visible in SPECT/CT images in the first 24 hours after injection and persisted until 7 days after injection and was validated by tissue counts of radioactivity. In contrast, MRI was unable to demonstrate targeted cardiac localization of MSCs in part because of the lower sensitivity of MRI. Noninvasive radionuclide imaging is well suited to dynamically track the biodistribution and trafficking of mesenchymal stem cells to both target and nontarget organs.

Paracrine and differentiation mechanisms underlying stem cell therapy for the damaged kidney

NATIVE KIDNEY ACUTE RENAL FAILURE (ARF) occurs in 2‐5% of hospitalized patients and is associated with high mortality (11). Allograft ARF occurs in 30‐50% of deceased donor kidney transplants and leads to longer hospital stays, a higher incidence of acute rejection, and reduced long-term graft survival (13). There is no specific therapy for ARF except for supportive care. A surge of evidence over the last decade supports an important role for inflammatory mediators, microvascular dysfunction, and apoptosis in the pathogenesis of the injury and extension phase of ARF (12). The mechanisms of recovery, less well understood, are felt to include a recapitulation of mechanisms originally involved in renal development. It is commonly believed that kidney progenitor cells, from either the kidney or an extrarenal source, repopulate the kidney during the recovery phase of ARF and drive the repair process (9). A popular model was that damaged/dead cells are removed and the kidney stem cells migrate to necrotic areas, differentiate, and repopulate the kidney. Indeed, injured human kidney transplants from female donors into male recipients were found to have Y chromosome staining of tubular epithelial cells, demonstrating that they were of recipient origin (5). In support of this model are studies in mice in which labeled bone marrow was tracked with -galactosidase (-gal) staining into postischemic kidney. -Gal staining was observed in 20% of the proximal tubular cells at 1 wk after ischemia, and also to some extent within 48 h of ischemia (7). Despite the focus on stem cell effects on repair, mice without functional bone marrow in that study had a worse early course of ARF, suggesting that bone marrow-derived cells had an early protective effect on the injury process. In another murine study, male-derived cells were found in female mouse kidney recipients after ischemia (8). However, recent work has shown that -gal staining to identify differentiated stem cells has considerable limitations, false positive results can result from the kidney’s endogenous -gal, and the staining validity is vulnerable to slight pH shifts (3). In a cisplatin model of ARF, administration of mesenchymal stem cells improved kidney function and structure (10). Given that Y chromosome-containing cells were found in recipient tubular epithelium of female mice and expressed specific lectin-binding proteins found in proximal tubules, transdifferentiation was concluded to be the mechanism of enhanced repair. Administration of skeletal muscle-derived stem cells that were differentiated into endothelial cells provided early protection from ischemic ARF in mice and were detected in glomeruli and peritubular capillaries using green fluorescent protein detection (1).

Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis.

Strategies for genetic prenatal diagnosis on fetal cells in the maternal circulation have been limited by lack of a cell type present only in fetal blood. However, the recent identification of mesenchymal stem cells (MSC) in first trimester fetal blood offers the prospect of targeting MSC for non-invasive prenatal diagnosis. We developed protocols for fetal MSC enrichment from maternal blood and determined sensitivity and specificity in mixing experiments of male fetal MSC added to female blood, in dilutions from 1 in 10(5) to 10(8). We then used the optimal protocol to isolate fetal MSC from maternal blood in the first trimester, using blood taken after surgical termination of pregnancy as a model of increased feto-maternal haemorrhage. In model mixtures, we could amplify one male fetal MSC in 2.5 x 10(7) adult female nucleated cells, yielding a 100% pure population of fetal cells, but not one fetal MSC in 10(8) nucleated cells. Fetal MSC were identified in one of 20 post-termination maternal blood samples and confirmed as fetal MSC by XY fluorescence in-situ hybridization (FISH), immunophenotyping and osteogenic and adipogenic differentiation. We report the isolation of fetal MSC from maternal blood; however, their rarity in post-termination blood suggests they are unlikely to have a role in non-invasive prenatal diagnosis. Failure to locate these cells routinely may be attributed to their low frequency in maternal blood, to sensitivity limitations of enrichment technology, and/or to their engraftment in maternal tissues soon after transplacental passage. We speculate that gender microchimerism in post-reproductive maternal tissues might result from feto-maternal trafficking of MSC in early pregnancy.