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Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymal stem/stromal cells
Abstract and Figures
Background
Fetal mesenchymal stem/stromal cells (MSC) represent a developmentally-advantageous cell type with translational potential.
To enhance adult MSC migration, studies have focussed on the role of the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12), but more recent work implicates an intricate system of CXCR4 receptor dimerization, intracellular localization, multiple ligands, splice variants and nuclear accumulation. We investigated the intracellular localization of CXCR4 in fetal bone marrow-derived MSC and role of intracellular trafficking in CXCR4 surface expression and function.
Results
We found that up to 4% of human fetal MSC have detectable surface-localized CXCR4. In the majority of cells, CXCR4 is located not at the cell surface, as would be required for ‘sensing’ migratory cues, but intracellularly. CXCR4 was identified in early endosomes, recycling endosomes, and lysosomes, indicating only a small percentage of CXCR4 travelling to the plasma membrane. Notably CXCR4 was also found in and around the nucleus, as detected with an anti-CXCR4 antibody directed specifically against CXCR4 isoform 2 differing only in N-terminal sequence. After demonstrating that endocytosis of CXCR4 is largely independent of endogenously-produced SDF-1, we next applied the cytoskeletal inhibitors blebbistatin and dynasore to inhibit endocytotic recycling. These increased the number of cells expressing surface CXCR4 by 10 and 5 fold respectively, and enhanced the number of cells migrating to SDF1 in vitro (up to 2.6 fold). These molecules had a transient effect on cell morphology and adhesion, which abated after the removal of the inhibitors, and did not alter functional stem cell properties.
Conclusions
We conclude that constitutive endocytosis is implicated in the regulation of CXCR4 membrane expression, and suggest a novel pharmacological strategy to enhance migration of systemically-transplanted cells.
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... As infection progresses, co-receptor tropism changes can occur, increasing the frequency of CXCR4 utilisation [152]. Expression of CD4, CXCR4 and CCR5 mRNA by MSCs has been observed in vitro [101,102,111,117,[153][154][155][156][157][158][159][160][161]]. However, the reported level of expression and localisation of these receptors/co-receptors is inconsistent. ...
... SDF-1-induced chemokine signals increase expression of CXCR4 on the surface of murine MSCs [158]. Upon binding SDF-1, MSC CXCR4 expression is decreased, and the co-receptor is internalised through endocytosis or micropinocytosis [157,175]. CXCR4 is then internally degraded by lysosomes or is recycled back to the cell surface [175,176]. ...
The introduction of antiretroviral therapy (ART) and highly active antiretroviral therapy (HAART) has transformed human immunodeficiency virus (HIV)-1 into a chronic, well-managed disease. However, these therapies do not eliminate all infected cells from the body despite suppressing viral load. Viral rebound is largely due to the presence of cellular reservoirs which support long-term persistence of HIV-1. A thorough understanding of the HIV-1 reservoir will facilitate the development of new strategies leading to its detection, reduction, and elimination, ultimately leading to curative therapies for HIV-1. Although immune cells derived from lymphoid and myeloid progenitors have been thoroughly studied as HIV-1 reservoirs, few studies have examined whether mesenchymal stromal/stem cells (MSCs) can assume this function. In this review, we evaluate published studies which have assessed whether MSCs contribute to the HIV-1 reservoir. MSCs have been found to express the receptors and co-receptors required for HIV-1 entry, albeit at levels of expression and receptor localisation that vary considerably between studies. Exposure to HIV-1 and HIV-1 proteins alters MSC properties in vitro, including their proliferation capacity and differentiation potential. However, in vitro and in vivo experiments investigating whether MSCs can become infected with and harbour latent integrated proviral DNA are lacking. In conclusion, MSCs appear to have the potential to contribute to the HIV-1 reservoir. However, further studies are needed using techniques such as those used to prove that cluster of differentiation (CD)4 ⁺ T cells constitute an HIV-1 reservoir before a reservoir function can definitively be ascribed to MSCs.
Graphical abstract
MSCs may contribute to HIV-1 persistence in vivo in the vasculature, adipose tissue, and bone marrow by being a reservoir for latent HIV-1. To harbour latent HIV-1, MSCs must express HIV-1 entry markers, and show evidence of productive or latent HIV-1 infection. The effect of HIV-1 or HIV-1 proteins on MSC properties may also be indicative of HIV-1 infection.
... Nonetheless, these results are supported by the cellular localization of CXCR4, differing according to cell type, tumor histology, and tumor stage. Sarcomas derive from non-transformed mesenchymal stem cells [58] that express CXCR4 in the nucleus [59]. Normal epithelial cells or tissues and epithelial-derived primary tumors express CXCR4 in the membrane [60]. ...
Poor long-term survival in localized high-risk soft tissue sarcomas (STSs) of the extremities and trunk highlights the need to identify new prognostic factors. CXCR4 is a chemokine receptor involved in tumor progression, angiogenesis, and metastasis. The aim of this study was to evaluate the association between CXCR4 expression in tumor tissue and survival in STSs patients treated with neoadjuvant therapy. CXCR4 expression was retrospectively determined by immunohistochemical analysis in serial specimens including initial biopsies, tumors post-neoadjuvant treatment, and tumors after relapse. We found that a positive cytoplasmatic expression of CXCR4 in tumors after neoadjuvant treatment was a predictor of poor recurrence-free survival (RFS) (p = 0.003) and overall survival (p = 0.019) in synovial sarcomas. We also found that positive nuclear CXCR4 expression in the initial biopsies was associated with poor RFS (p = 0.022) in undifferentiated pleomorphic sarcomas. In conclusion, our study adds to the evidence that CXCR4 expression in tumor tissue is a promising prognostic factor for STSs.
... In fact, our expression patterns are consistent with what is known about G-protein coupled receptors, which undergo internalization after interaction with ligands [49]. Additionally, ligand-induced endocytosis of CXCR4 and its internal sequestration are also well known in leukocytes, stem cells, and tumor cells [50][51][52]. ...
Despite recent therapeutic advances, pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease with limited therapeutic options. Immune checkpoint inhibitors (ICIs) have demonstrated promising results in many cancers, but thus far have yielded little clinical benefit in PDAC. Based on recent combined targeting of programmed cell death protein-1 (PD-1) and C-X-C chemokine receptor 4 (CXCR4) in patient-derived xenografts (PDXs) and a pilot clinical trial, we sought to elucidate potential interactions between PD-1 and CXCR4. We observed concomitant expression and direct interaction of PD-1 and CXCR4 in PDAC cells. This interaction was disrupted upon CXCR4 antagonism with AMD3100 and led to increased cell surface expression of PD-1. Importantly, CXCR4-mediated PDAC cell migration was also blocked by PD-1 inhibition. Our work provides a possible mechanism by which prior studies have demonstrated that combined CXCR4 and PD-1 inhibition leads to decreased tumor growth. This is the first report investigating PD-1 and CXCR4 interactions in PDAC cells and our results can serve as the basis for further investigation of combined therapeutic targeting of CXCR4 and PD-1.
... There was also CXCR4 in the cell nucleus. The treatment that increases the expression of CXCR4 in the membrane turns out to increase metastasis 2.6 times (Pelekanos et al., 2014). ...
Objective:
Investigate the effect of SDF1a, nuclear, and cytoplasmic CXCR4 breast cancer tissue on metastasis and overall survival in patients with complete-chemotherapy and no-chemotherapy.
Methods:
Cohort ambidirectional design was employed with survival analysis that followed the patient's diagnosis until obtaining the outcome, distant metastasis, or death. We analyzed samples in three groups (all-patient, no-chemotherapy, and complete-chemotherapy groups). Breast cancer cell nuclear and cytoplasm expressions of CXCR4 protein were examined using immunohistochemistry. Amplification of mRNA SDF1a of breast cancer tissue was examined using rtPCR on 131 samples from the same initial paraffin block.
Results:
In the distant metastasis and Overall Survival (OS) analysis, there was no correlation between cytoplasmic and nuclear CXCR4 in all-patient, no-chemotherapy, and complete-chemotherapy groups. SDF1a was significantly correlated to shorter distant metastasis and poor OS in the all-patient (p=0.004 and p=0.04, respectively) and no-chemotherapy group (p=0.008 and p=0.026, respectively). However, in the complete-chemotherapy group, SDF1a was not correlated to either metastasis (p=0.527) or OS (p=0.993), advanced stage demonstrated a strong association on shorter distant metastatic in no-chemotherapy (p=0.021) and complete-chemotherapy group (p=0.004) and also poor OS in both groups (p=0.006 and p=0.002, respectively). The hormone receptor showed a protective effect on the no-chemotherapy group's OS (p= 0.019). Meanwhile, not undergoing chemotherapy was associated with poor OS in the all-patient group (p= 0.011).
Conclusion:
SDF1a mRNA amplification has a significant correlation with the occurrence of metastasis and OS in all-patient and no-chemotherapy group. Undergoing chemotherapy negates the effect of SDF1a for distant metastasis and OS.
... Such flexibility is largely contributed by the stimulation of diverse G-protein-dependent and -independent pathways and the notable expression mechanism of CXCR4 characterized by agile endocytosis and recycling patterns. The cellular CXCR4 proteins, which are predominantly localized in intracellular cytoplasm, are partially-to-mostly expressed at the surface membrane upon stimulation by CXCL12 [4]. ...
CXC-chemokine receptor type 4 (CXCR4), a 7-transmembrane receptor family member, displays multifaceted roles, participating in immune cell migration, angiogenesis, and even adipocyte metabolism. However, the activity of such a ubiquitously expressed receptor in epithelial gland organogenesis has not yet been fully explored. To investigate the relationship between CXCL12/CXCR4 signaling and embryonic glandular organogenesis, we used an ex vivo culture system with live imaging and RNA sequencing to elucidate the transcriptome and protein-level signatures of AMD3100, a potent abrogating reagent of the CXCR4-CXCL12 axis, imprinted on the developing organs. Immunostaining results showed that CXCR4 was highly expressed in embryonic submandibular gland, lung, and pancreas, especially at the periphery of end buds containing numerous embryonic stem/progenitor cells. Despite no significant increase in apoptosis, AMD3100-treated epithelial organs showed a retarded growth with significantly slower branching and expansion. Further analyses with submandibular glands revealed that such responses resulted from the AMD3100-induced precocious differentiation of embryonic epithelial cells, losing mitotic activity. RNA sequencing analysis revealed that inhibition of CXCR4 significantly down-regulated polycomb repressive complex (PRC) components, known as regulators of DNA methylation. Treatment with PRC inhibitor recapitulated the AMD3100-induced precocious differentiation. Our results indicate that the epigenetic modulation by the PRC-CXCR12/CXCR4 signaling axis is crucial for the spatiotemporal regulation of proliferation and differentiation of embryonic epithelial cells during embryonic glandular organogenesis.
Intracellular protein abundance is routinely measured in mammalian cells using population-based techniques such as western blotting which fail to capture single cell protein levels or using fluorescence microscopy which is although suitable for single cell protein detection but not for rapid analysis of large no. of cells. Flow cytometry offers rapid, high-throughput, multiparameter-based analysis of intracellular protein expression in statistically significant no. of cells at single cell resolution. In past few decades, customized assays have been developed for flow cytometric detection of specific intracellular proteins. This review discusses the scope of flow cytometry for intracellular protein detection in mammalian cells along with specific applications. Technological advancements to overcome the limitations of traditional flow cytometry for the same are also discussed.
Mesenchymal stem cells (mesenchymal stromal cells, MSC) are multipotent stem cells that can differentiate into cells of at least three mesodermal lineages, namely adipocytes, osteoblasts, and chondrocytes, and have potent immunomodulatory properties. Epigenetic modifications are critical regulators of gene expression and cellular differentiation of mesenchymal stem cells (MSCs). Epigenetic machinery controls MSC differentiation through direct modifications to DNA and histones. Understanding the role of epigenetic machinery in MSC is crucial for the development of effective cell-based therapies for degenerative and inflammatory diseases. In this review, we summarize the current understanding of the role of epigenetic control of MSC differentiation and immunomodulatory properties.
Cell Death and Disease (2023) 14:720 ; https://doi.org/10.1038/s41419-023-06239-4
After ischemia, cells in the brain parenchyma upregulate stromal derived factor 1 (SDF1), driving chemokine receptor CXCR4-mediated migration of adult neural stem cells to the ischemic injury. We discovered a novel regulator of CXCR4 in neural stem cells, low-density lipoprotein receptor related protein 1 (LRP1). We employed Nestin-driven knockout of LRP1 and induction of td-tomato in neural stem cells of adult mice. We observed reduced localization of td-tomato positive cells to the lesion, and find disrupted CXCR4-mediated neural stem cell migration in vitro, which is likely driven by LRP1-mediated loss of CXCR4 expression in vivo. Our results suggest that LRP1 is a novel regulator of CXCR4 in neural stem cells. This heretofore unknown interaction between LRP1 and CXCR4 could have significant consequences to multiple aspects of neural stem cell physiology.
After ischemia, cells in the brain parenchyma upregulate stromal derived factor 1 (SDF1), driving chemokine receptor CXCR4-mediated migration of adult neural stem cells from the subventricular zone (SVZ) to the ischemic injury. We discovered a novel regulator of CXCR4 in neural stem cells, low-density lipoprotein receptor related protein 1 (LRP1). We employed a tamoxifen-inducible Nestin-Cre to drive expression of a tdTomato reporter and also knockout floxed LRP1 in adult mice and then subjected mice to middle-cerebral artery occlusion. Examination 2 weeks post-stroke reveals a loss of tdTomato positive cells localizing from the SVZ to the lesion. We show that loss of LRP1 disrupts CXCR4-mediated neural stem cell migration in vitro , which is likely driven by LRP1-mediated loss of CXCR4 expression in vivo . Altogether, our results suggest that LRP1 is a novel regulator of CXCR4 in neural stem cells.
Highlights
- LRP1 KO in adult neural stem cells disrupts migration to ischemic lesions in vivo .
- LRP1 KO in adult neural stem cells disrupts migration towards SDF1 in vitro .
- LRP1 positively regulates expression of CXCR4 in adult neural stem cells.
ETOC blurb
Adult neural stem cells can home to ischemic brain injury and are considered an important part of the repair process after stroke. However, little is known about what molecules help drive this response. The authors discovered that LRP1 is a novel regulator of CXCR4, which is essential for neural stem cell migration to ischemic injury.
Crosstalk between Kupffer cells (KCs) and hepatic stellate cells (HSCs) plays an important role in multiple liver disease conditions, including the formation of liver fibrosis in alcohol-associated liver disease (AALD). Therapeutic targeting of the KC-HSC crosstalk is a prime target for therapeutic interventions. Herein, a novel modular nanosystem was designed and prepared through the self-assembly utilizing boric acid and catechol interactions to prepare polymers modified with a CXCR4-inhibiting moieties. The polymers were used to encapsulate anti-miR-155 and to block the undesirable crosstalk between HSCs and KCs by downregulating miR-155 expression in KCs with the parallel inhibition of CXCR4 signaling in activated HSCs. The combined inhibition of miR-155 and CXCR4 at two different liver cell types achieved improved antifibrosis effects in a mouse model of AALD fibrosis. Our finding highlights the key role that blocking the undesirable crosstalk between HSCs and KCs plays in reversing AALD fibrosis as well as demonstrates a proof-of-concept approach for designing and constructing multifunctional delivery nanosystems using orthogonal functional modules based on the understanding of disease mechanisms.
The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs.
The ability to interact with cell-surface glycosaminoglycans (GAGs) is essential to the cell migration properties of chemokines, but association with soluble GAGs induces the oligomerization of most chemokines including CXCL12. Monomeric CXCL12, but not dimeric CXCL12, is cardioprotective in a number of experimental models of cardiac ischemia. We found that co-administration of heparin, a common treatment for myocardial infarction, abrogated the protective effect of CXCL12 in an ex vivo rat heart model for myocardial infarction. The interaction between CXCL12 and heparin oligosaccharides has previously been analyzed through mutagenesis, in vitro binding assays, and molecular modeling. However, complications from heparin-induced CXCL12 oligomerization and studies using very short oligosaccharides have led to inconsistent conclusions as to the residues involved, the orientation of the binding site, and whether it overlaps with the CXCR4 N- terminal site. We used a constitutively dimeric variant to simplify the NMR analysis of CXCL12 binding heparin oligosaccharides of varying length. Biophysical and mutagenic analyses reveal a CXCL12/heparin interaction surface that lies perpendicular to the dimer interface, does not involve the chemokine aminoterminus, and partially overlaps with the CXCR4 binding site. We further demonstrate that heparin-mediated enzymatic protection results from the promotion of dimerization rather than direct heparin binding to the CXCL12 aminoterminus. These results clarify the structural basis for GAG recognition by CXCL12 and lend insight into the development of CXCL12-based therapeutics.
Chemokines and their receptors are implicated in formation of colorectal cancer metastases. Especially CXCR4 is an important
factor, determining migration, invasiveness, metastasis and proliferation of colorectal cancer cells. Object of this study
was to determine expression of CXCR4 in tumor tissue of colorectal cancer patients and associate CXCR4 expression levels to
clinicopathological parameters. Levels of CXCR4 expression of a random cohort of patients, who underwent primary curative
resection of a colorectal carcinoma, were retrospectively determined by quantitative real-time RT-PCR and semi-quantitative
analyses of immunohistochemical stained paraffin sections. Expression levels were associated to clinicopathological parameters.
Using RT-PCR we found that a high expression of CXCR4 in the primary tumor was an independent prognostic factor for a poor
disease free survival (p = 0.03, HR: 2.0, CI = 1.1–3.7). Immunohistochemical staining showed that nuclear distribution of CXCR4 in the tumor cells
was inversely associated with disease free and overall survival (p = 0.04, HR: 2.6, CI = 1.0–6.2), while expression in the cytoplasm was not associated with prognosis. In conclusion, our study
showed that a high expression of nuclear localized CXCR4 in tumor cells is an independent predictor for poor survival for
colorectal cancer patients.
Cell migration involves complex physical and chemical interactions with the substrate. To probe the mechanical interactions under different regions of migrating 3T3 fibroblasts, we have disrupted cell-substrate adhesions by local application of the GRGDTP peptide, while imaging stress distribution on the substrate with traction force microscopy. Both spontaneous and GRGDTP-induced detachment of the trailing edge caused extensive cell shortening, without changing the overall level of traction forces or the direction of migration. In contrast, disruption of frontal adhesions caused dramatic, global loss of traction forces before any significant shortening of the cell. Although traction forces and cell migration recovered within 10-20 min of transient frontal treatment, persistent treatment with GRGDTP caused the cell to develop traction forces elsewhere and reorient toward a new direction. We conclude that contractile forces of a fibroblast are transmitted to the substrate through two distinct types of adhesions. Leading edge adhesions are unique in their ability to transmit active propulsive forces. Their functions cannot be transferred directly to existing adhesions upon detachment. Trailing end adhesions create passive resistance during cell migration and readily redistribute their loads upon detachment. Our results indicate the distinct nature of mechanical interactions at the leading versus trailing edges, which together generate the mechanical interactions for fibroblast migration.
Mesenchymal stem cells (MSCs) can be used as a delivery vehicle for gene therapy agamst brain tumors, because these cells have a migratory capacity toward glioma cells. Soluble factors including chemokines or growth factors expressed and released by glioma cells mediate the tropism of MSCs for gliomas. Among them, stromal cell-derived factor-1α (SDF-1α) has been identified as a key molecule related to the tropism of MSC in many cancers containing gliomas. In this study, we found that overexpression of the SDF-la receptor, CXCR4, on human umbilical cord blood-derived MSCs (hUCB-MSCs) enhanced the migratory capacity of MSCs toward gliomas. We showed that hUCB-MSCs have the migration ability toward the glioma cell lines and primary glioma cells. SDF-1α treatment increased the migration capacity of hUCB-MSCs in a dose-dependent manner and inhibition of SDF-1α or CXCR4 by treatment with the anti-SDF-1α or the CXCR4 antagonist AMD3100 blocked the migration capacity of hUCB-MSCs toward glioma cells. Furthermore, CXCR4-overexpressed hUCB-MSCs (hMSCs-CXCR4) showed a stronger migration capacity toward glioma cells in vitro compared with control MSCs, and also exhibited enhanced migration to glioma cells in an intracranial human malignant glioma xenograft model. These results indicate that SDF-1α/CXCR4 could be involved in recruitment of hUCB-MSCs to glioma cells and that overexpression of CXCR4 may be a useful tool for stem cell-based glioma therapy.
Human mesenchymal stem/progenitor cells (MSCs) have been identified in adult bone marrow, but little is known about their presence during fetal life. MSCs were isolated and characterized in first-trimester fetal blood, liver, and bone marrow. When 106 fetal blood nucleated cells (median gestational age, 10+2 weeks [10 weeks, 2 days]) were cultured in 10% fetal bovine serum, the mean number (± SEM) of adherent fibroblastlike colonies was 8.2 ± 0.6/106 nucleated cells (69.6 ± 10/μL fetal blood). Frequency declined with advancing gestation. Fetal blood MSCs could be expanded for at least 20 passages with a mean cumulative population doubling of 50.3 ± 4.5. In their undifferentiated state, fetal blood MSCs were CD29+, CD44+, SH2+, SH3+, and SH4+; produced prolyl-4-hydroxylase, α-smooth muscle actin, fibronectin, laminin, and vimentin; and were CD45−, CD34−, CD14−, CD68−, vWF−, and HLA-DR−. Fetal blood MSCs cultured in adipogenic, osteogenic, or chondrogenic media differentiated, respectively, into adipocytes, osteocytes, and chondrocytes. Fetal blood MSCs supported the proliferation and differentiation of cord blood CD34+cells in long-term culture. MSCs were also detected in first-trimester fetal liver (11.3 ± 2.0/106 nucleated cells) and bone marrow (12.6 ± 3.6/106 nucleated cells). Their morphology, growth kinetics, and immunophenotype were comparable to those of fetal blood-derived MSCs and similarly differentiated along adipogenic, osteogenic, and chondrogenic lineages, even after sorting and expansion of a single mesenchymal cell. MSCs similar to those derived from adult bone marrow, fetal liver, and fetal bone marrow circulate in first-trimester human blood and may provide novel targets for in utero cellular and gene therapy.
Background aims:
The interaction between stromal cell-derived factor (SDF)-1 and its receptor CXCR4 is one of the mechanisms by which mesenchymal stromal cells (MSCs) are recruited to sites of injury. SDF-1 is upregulated in damaged tissues, but because the surface expression of CXCR4 on cultured MSCs is low, we investigated whether the delivery of CXCR4 into MSCs with the use of the cationic liposomal reagent IBAfect would increase their migration toward SDF-1.
Methods:
We examined (i) the effect of MSC confluency, passage number, duration of transfection and amount of IBAfect and plasmid on transfection efficiency as determined by flow cytometric analysis of CXCR4 and (ii) whether IBAfect-mediated CXCR4 transfection affected the viability, proliferation and differentiation of MSCs as well as their response toward an SDF-1 gradient in a trans-Matrigel migration assay.
Results:
We found that transfection efficiency of up to 40% was achieved after 24-h transfection of 50% confluent MSCs (at passage 4) with an IBAfect:plasmid ratio of 3.6 μL:0.6 μg, and CXCR4 transcript expression in transfected MSCs was 10(5)-fold higher than in non-transfected cells. Transfected MSCs retained their ability to differentiate to osteocytes and chondrocytes but had lower proliferation. Importantly, overexpression of surface CXCR4 with the use of IBAfect significantly increased (>3-fold) the number of cells migrating toward an SDF-1 gradient relative to cells migrating to media alone, compared with non-transfected cells (1.3-fold).
Conclusions:
Our results suggest that IBAfect-mediated delivery of CXCR4 into MSCs is a highly efficient technique that may be useful for enhancing the recruitment of systemically infused MSCs for tissue repair.
Stem cells have considerable potential to repair damaged organs and tissues. We previously showed that prenatal transplantation of human first trimester fetal blood mesenchymal stem cells (hfMSCs) in a mouse model of osteogenesis imperfecta (oim mice) led to a phenotypic improvement, with a marked decrease in fracture rate. Donor cells differentiated into mature osteoblasts, producing bone proteins and minerals, including collagen type Iα2, which is absent in nontransplanted mice. This led to modifications of the bone matrix and subsequent decrease of bone brittleness, indicating that grafted cells directly contribute to improvement of bone mechanical properties. Nevertheless, the therapeutic effect was incomplete, attributing to the limited level of engraftment in bone. In this study, we show that although migration of hfMSCs to bone and bone marrow is CXCR4-SDF1 (SDF1 is stromal-derived factor) dependent, only a small number of cells present CXCR4 on the cell surface despite high levels of internal CXCR4. Priming with SDF1, however, upregulates CXCR4 to increase the CXCR4(+) cell fraction, improving chemotaxis in vitro and enhancing engraftment in vivo at least threefold in both oim and wild-type bone and bone marrow. Higher engraftment in oim bones was associated with decreased bone brittleness. This strategy represents a step to improve the therapeutic benefits of fetal cell therapy toward being curative.
Chemokine CXCL12 is widely expressed in the central nervous system and essential for the proper functioning of human neural progenitor cells (hNPCs). Although CXCL12 is known to function through its receptor CXCR4, recent data have suggested that CXCL12 binds to chemokine receptor CXCR7 with higher affinity than to CXCR4. However, little is known about the function of CXCR7 in hNPCs. Using a primary hNPC culture system, we demonstrated that CXCL12 promotes hNPC survival in the events of camptothecin-induced apoptosis or growth factor deprivation, and that this effect requires both CXCR7 and CXCR4. Through fluorescence-activated cell sorting analysis and immunocytochemistry, we determined that CXCR7 is mainly localized in the early endosome, while CXCR4 is more broadly expressed at the cell surface and on both early and recycling endosomes. Furthermore, we found that endocytosis is required for the prosurvival function of CXCL12. Using dual-color total internal reflection fluorescence microscopy and immunoprecipitation, we demonstrated that CXCR7 quickly trafficks to plasma membrane in mediating CXCL12 endocytosis and colocalizes with CXCR4 after CXCL12 treatment. Investigating the molecular mechanisms, we found that ERK1/2 endocytotic signaling pathway is essential for hNPC survival upon apoptotic challenges. Consistent with these findings, a significantly higher number of apoptotic NPCs were found in the developing brain of CXCR7 knockout mice. In conclusion, CXCL12 protects hNPCs from apoptotic challenges through CXCR7- and CXCR4-mediated endocytotic signaling. Since survival of hNPCs is important for neurogenesis, CXCR7 may become a new therapeutic target to properly regulate critical processes of brain development. STEM CELLS2012;30:2571-2583.