Mesenchymal stem cells (MSC) are multipotent progenitor cells localized in the stromal compartment of the bone marrow (BM). The potential of MSC for mesenchymal differentiation has been well documented in different animal models predominantly on rodents. However, information regarding bovine MSC (bMSC) is limited, and the differentiation potential of bMSC derived from fetal BM remains unknown. In the present study we sought to isolate bMSC from abattoir-derived fetal BM and to characterize the multipotent and differentiation potential under osteogenic, chondrogenic and adipogenic conditions by quantitative and qualitative analyses.
Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. These cells expressed high levels of MSC surface markers (CD73, CD90, and CD105) and low levels of hematopoietic surface markers (CD34 and CD45). Culture of bMSC under osteogenic conditions during a 27-day period induced up-regulation of the osteocalcin (OC) gene expression and alkaline phosphatase (ALPL) activity, and promoted mineralization of the matrix. Increasing supplementation levels of ascorbic acid to culture media enhanced osteogenic differentiation of bMSC; whereas, reduction of FBS supplementation compromised osteogenesis. bMSC increased expression of cartilage-specific genes aggrecan (ACAN), collagen 2A1 (COL2A1) and SRY (sex-determining region Y) box 9 (SOX9) at Day 21 of chondrogenic differentiation. Treatment of bMSC with adipogenic factors increased levels of fatty acid-binding protein 2 (AP2) mRNA and accumulation of lipid vacuoles after 18 days of culture. NANOG mRNA levels in differentiating bMSC were not affected during adipogenic culture; however, osteogenic and chondrogenic conditions induced higher and lower levels, respectively.
Our analyses revealed the potential multilineage differentiation of bMSC isolated from abattoir-derived fetal BM. NANOG mRNA pattern in differentiating bMSC varied according to differentiation culture conditions. The osteogenic differentiation of bMSC was affected by ascorbic acid and FBS concentrations in culture media. The simplicity of isolation and the differentiation potential suggest that bMSC from abattoir-derived fetal BM are appropriate candidate for investigating MSC biology and for eventual applications for regenerative therapy.
Mesenchymal stem cell; Bovine fetuses; Differentiation potential; Multipotency
Mesenchymal stem cells (MSCs) have received great attention due to their remarkable regenerative, angiogenic, antiapoptotic, and immunosuppressive properties. Although conventionally isolated from the bone marrow, they are known to exist in all tissues and organs, raising the question on whether they are identical cell populations or have important differences at the molecular level. To better understand the relationship between MSCs residing in different tissues, we analyzed the expression of genes related to pluripotency (SOX2 and OCT-4) and to adipogenic (C/EBP and ADIPOR1), osteogenic (OMD and ALP), and chondrogenic (COL10A1 and TRPV4) differentiation in cultures derived from murine endodermal (lung) and mesodermal (adipose) tissue maintained in different conditions. MSCs were isolated from lungs (L-MSCs) and inguinal adipose tissue (A-MSCs) and cultured in normal conditions, in overconfluence or in inductive medium for osteogenic, adipogenic, or chondrogenic differentiation. Cultures were characterized for morphology, immunophenotype, and by quantitative real-time reverse transcription–polymerase chain reaction for expression of pluripotency genes or markers of differentiation. Bone marrow–derived MSCs were also analyzed for comparison of these parameters. L-MSCs and A-MSCs exhibited the typical morphology, immunophenotype, and proliferation and differentiation pattern of MSCs. The analysis of gene expression showed a higher potential of adipose tissue–derived MSCs toward the osteogenic pathway and of lung-derived MSCs to chondrogenic differentiation, representing an important contribution for the definition of the type of cell to be used in clinical trials of cell therapy and tissue engineering.
Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.
Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are the two widely studied and characterized adult stem cells. Thus far, MSCs were obtained from the bone marrow, which is a painful procedure. Therefore, MSCs from less common sources like cord blood, adipose tissue, tooth pulp, and so on, have been the subject of research. The purpose of this study is to explore the possibility of finding MSCs from a less controversial, easy, and abundant source, such as the umbilical cord, for potential regenerative medicine applications.
Study Design and Methods:
Five bone marrow samples (BM), seventy cord blood units (CB), and four umbilical cord matrix (CM) samples have been used for the study. Expanded MSCs were checked for biomarker expression by flow cytometry and were also checked for their differentiation to mesodermal and ectodermal lineages.
MSCs could be isolated from 100% BM and CM samples, as compared to only 6% of CB samples. The fold expansion of the mesenchymal stem cells observed in CB (CB-MSCs) was distinctly higher as compared to BM (BM-MSCs) and CM (CM-MSCs). MSCs isolated from all the three sources expressed a characteristic mesenchymal phenotype of CD45 − /vWF − /CD14 − /CD31 − /CD73 + /CD105 + /SSEA4 + /CD29 + /CD44 + /HLAABC +, whereas, the HLA DR was conspicuously absent in CM-MSCs and CB-MSCs. Although osteogenic, chondrogenic, and neural differentiation was observed in MSCs from all sources, adipogenic differentiation was observed only in BM-MSCs.
CM-MSCs are a dependable source of an unlimited number of MSCs for autologous and allogenic use in regenerative medicine.
Mesenchymal stem cells; bone marrow; umbilical cord blood; umbilical cord
Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several mesenchymal lineages, classically derived from bone marrow (BM) but potentially from umbilical cord blood (UCB). Although they are becoming a good tool for regenerative medicine, they usually need to be expanded in fetal bovine serum (FBS)-supplemented media. Human platelet lysate (HPL) has recently been proposed as substitute for safety reasons, but it is not yet clear how this supplement influences the properties of expanded MSCs.
In the present study, we compared the effect of various media combining autologous HPL with or without FBS on phenotypic, proliferative and functional (differentiation, cytokine secretion profile) characteristics of human BM-derived MSCs.
Despite less expression of adipogenic and osteogenic markers, MSCs cultured in HPL-supplemented media fully differentiated along osteoblastic, adipogenic, chondrogenic and vascular smooth muscle lineages. The analyses of particular specific proteins expressed during osteogenic differentiation (calcium-sensing receptor (CaSR) and parathormone receptor (PTHR)) showed their decrease at D0 before any induction for MSC cultured with HPL mostly at high percentage (10%HPL). The cytokine dosage showed a clear increase of proliferation capacity and interleukin (IL)-6 and IL-8 secretion.
This study shows that MSCs can be expanded in media supplemented with HPL that can totally replace FBS. HPL-supplemented media not only preserves their phenotype as well as their differentiation capacity, but also shortens culture time by increasing their growth rate.
Regulation of skeletal remodeling appears to influence the differentiation of multipotent mesenchymal stem cells (MSC) resident in the bone marrow. As murine marrow cultures are contaminated with hematopoietic cells, they are problematic for studying direct effects of mechanical input. Here we use a modified technique to isolate marrow-derived MSC (mdMSC) from adult mice, yielding a population able to differentiate into adipogenic and osteogenic phenotypes that is devoid of hematopoietic cells. In pure mdMSC populations, a daily strain regimen inhibited adipogenic differentiation, suppressing expression of PPARγ and adiponectin. Strain increased β-catenin and inhibition of adipogenesis required this effect. Under osteogenic conditions, strain activated β-catenin signaling and increased expression of WISP1 and COX2. mdMSC were also generated from mice lacking caveolin-1, a protein known to sequester β-catenin: caveolin-1(−/−) mdMSC exhibited retarded differentiation along both adipogenic and osteogenic lineages, but retained mechanical responses that involved β-catenin activation. Interestingly, caveolin-1(−/−) mdMSC failed to express bone sialoprotein and did not form mineralized nodules. In summary, mdMSC from adult mice respond to both soluble factors and mechanical input, with mechanical activation of β-catenin influencing phenotype. As such, these cells offer a useful model for studies of direct mechanical regulation of MSC differentiation and function.
stem cell; osteoblast; adipocyte; mechanical loading; caveolin-1
Alternative sources of mesenchymal stem cells (MSCs) for replacing bone marrow (BM) have been extensively investigated in the field of bone tissue engineering. The purpose of this study was to compare the osteogenic potential of canine MSCs derived from adipose tissue (AT), BM, umbilical cord blood (UCB), and Wharton's jelly (WJ) using in vitro culture techniques and in vivo orthotopic implantation assays. After canine MSCs were isolated from various tissues, the proliferation and osteogenic potential along with vascular endothelial growth factor (VEGF) production were measured and compared in vitro. For the in vivo assay, MSCs derived from each type of tissue were mixed with β-tricalcium phosphate and implanted into segmental bone defects in dogs. Among the different types of MSCs, AT-MSCs had a higher proliferation potential and BM-MSCs produced the most VEGF. AT-MSCs and UCB-MSCs showed greater in vitro osteogenic potential compared to the other cells. Radiographic and histological analyses showed that all tested MSCs had similar osteogenic capacities, and the level of new bone formation was much higher with implants containing MSCs than cell-free implants. These results indicate that AT-MSCs, UCB-MSCs, and WJ-MSCs can potentially be used in place of BM-MSCs for clinical bone engineering procedures.
cell source; dogs; mesenchymal stem cells; osteogenesis
REX1/ZFP42 is a well-known embryonic stem cell (ESC) marker. However, the role of REX1, itself, is relatively unknown because the function of REX1 has only been reported in the differentiation of ESCs via STAT signaling pathways. Human mesenchymal stem cells (hMSCs) isolated from young tissues and cancer cells express REX1.
Human umbilical cord blood-derived MSCs (hUCB-MSCs) and adipose tissue-derived MSCs (hAD-MSCs) strongly express REX1 and have a lower activation status of p38 MAPK, but bone marrow-derived MSCs (hBM-MSCs) have weak REX1 expression and higher activation of p38 MAPK. These results indicated that REX1 expression in hMSCs was positively correlated with proliferation rates but inversely correlated with the phosphorylation of p38 MAPK. In hUCB-MSCs, the roles of REX1 and p38 MAPK were investigated, and a knockdown study was performed using a lentiviral vector-based small hairpin RNA (shRNA). After REX1 knockdown, decreased cell proliferation was observed. In REX1 knocked-down hUCB-MSCs, the osteogenic differentiation ability deteriorated, but the adipogenic potential increased or was similar to that observed in the controls. The phosphorylation of p38 MAPK in hUCB-MSCs significantly increased after REX1 knockdown. After p38 MAPK inhibitor treatment, the cell growth in REX1 knocked-down hUCB-MSCs almost recovered, and the suppressed expression levels of CDK2 and CCND1 were also restored. The expression of MKK3, an upstream regulator of p38 MAPK, significantly increased in REX1 knocked-down hUCB-MSCs. The direct binding of REX1 to the MKK3 gene was confirmed by a chromatin immunoprecipitation (ChIP) assay.
These findings showed that REX1 regulates the proliferation/differentiation of hMSCs through the suppression of p38 MAPK signaling via the direct suppression of MKK3. Therefore, p38 MAPK and REX-1 status can determine the cell fate of adult stem cells (ASCs). These results were the first to show the role of REX1 in the proliferation/differentiation of ASCs.
Background & objectives:
Mesencymal stem cells (MSCs) derived from foetal tissues present a multipotent progenitor cell source for application in tissue engineering and regenerative medicine. The present study was carried out to derive foetal mesenchymal stem cells from ovine source and analyze their differentiation to osteogenic linage to serve as an animal model to predict human applications.
Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR) analysis and confirmed by sequencing using genetic analyzer.
Ovine foetal samples were processed to obtain mononuclear (MNC) cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45-/CD14- was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinβ1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP) activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells.
Interpretation & conclusions:
The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established. Criteria proposed for defining MSCs by this study includes the cell adherence to culture plates, specific surface protein profiles and differentiation to osteogenic lineage. The MSCs and osteogenic differentiated cells in this ovine animal model may serve as a large source for stem cell applications in regenerative medical therapies.
Bone marrow; characterization; differentiation; foetal; MACS; mesenchymal stem cells; multilineage; multipotent; osteogenic
Mesenchymal stem cell (MSC) therapy is a promising approach to promote tissue regeneration by either differentiating the MSCs into the desired cell type or by using their trophic functions to promote endogenous tissue repair. These strategies of regenerative medicine are limited by the availability of MSCs at the point of clinical care. Our laboratory has recently identified multipotent mesenchymal progenitor cells (MPCs) in traumatically injured muscle tissue, and the objective of this study was to compare these cells to a typical population of bone marrow-derived MSCs. Our hypothesis was that the MPCs exhibit multilineage differentiation and expression of trophic properties that make functionally them equivalent to bone marrow-derived MSCs for tissue regeneration therapies. Quantitative evaluation of their proliferation, metabolic activity, expression of characteristic cell-surface markers and baseline gene expression profile demonstrate substantial similarity between the two cell types. The MPCs were capable of differentiation into osteoblasts, adipocytes and chondrocytes, but they appeared to demonstrate limited lineage commitment compared to the bone-marrow derived MSCs. The MPCs also exhibited trophic (i.e., immunoregulatory and pro-angiogenic) properties that were comparable to those of MSCs. These results suggest that the traumatized muscle-derived MPCs may not be a direct substitute for bone marrow-derived MSCs. However, because of their availability and abundance, particularly following orthopaedic injuries when traumatized muscle is available to harvest autologous cells, MPCs are a promising cell source for regenerative medicine therapies designed to take advantage of their trophic properties.
Mesenchymal stem cells; progenitor cells; tissue engineering; regenerative medicine; immunoregulation; angiogenesis
Adipose-derived stem cells (ADSCs) are mesenchymal stem cells (MSCs) that can be extracted from adipose tissue and obtained by a less invasive method and in larger quantities compared with bone marrow–derived MSCs. The objective of this study was to harvest ADSCs from piglets and to explore their neuronal differentiation potential.
Adipose tissue from piglet facial or abdominal fat was digested with collagenase type XI, followed by filter and centrifugation; the isolated adipose stromal cells were cultured in dishes. MSC markers were measured by flow cytometry; 2 to 5 passage cells were used for in vitro differentiation. Adipogenic, chondrogenic, osteogenic, and neuronal differentiation was induced by incubation of the ADSCs with different induction media.
ADSCs were easily expanded to beyond 15 passages, maintaining the undifferentiated state and exhibiting MSC characteristics and markers CD29, CD44, and CD90. ADSCs differentiated into other mesodermal cells including adipocytes, chondrocytes, and osteocytes. These cells were induced to differentiate into neuron-like cells as evidenced by neuronal morphology and the presence of neuronal markers including microtubule-associated protein 2, neuronal nuclear antigen, and β-tubulin III.
ADSCs can be readily obtained from a small amount fat tissue and expanded in culture. Adipose tissue may be an alternative source of stem cell therapy for nervous system injury.
Mesenchymal stem cell; Adipose tissue; Neuronal differentiation
This study developed and tested a serum-free and xeno-free culture medium (SFM-XF) using human bone marrow- and adipose-derived mesenchymal stem cells (MSCs). Human MSCs expanded in SFM-XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of traditional serum-containing culture medium (SCM). The SFM-XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to SCM for the production of large scale, functionally competent MSCs for future clinical applications.
Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, cartilage damage, and cardiac diseases. Despite remarkable progress in clinical trials, most applications still use traditional culture media containing fetal bovine serum or serum-free media that contain serum albumin, insulin, and transferrin. The ill-defined and variable nature of traditional culture media remains a challenge and has created a need for better defined xeno-free culture media to meet the regulatory and long-term safety requirements for cell-based therapies. We developed and tested a serum-free and xeno-free culture medium (SFM-XF) using human bone marrow- and adipose-derived MSCs by investigating primary cell isolation, multiple passage expansion, mesoderm differentiation, cellular phenotype, and gene expression analysis, which are critical for complying with translation to cell therapy. Human MSCs expanded in SFM-XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of MSCs expanded in traditional serum-containing culture medium (SCM). To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded in SFM-XF and SCM were compared, revealing relatively similar expression profiles. In addition, the SFM-XF supported the isolation and propagation of human MSCs from primary human marrow aspirates, ensuring that these methods and reagents are compatible for translation to therapy. The SFM-XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to serum-containing media for the production of large scale, functionally competent MSCs for future clinical applications.
Bone marrow stromal cells; Cellular therapy; Chondrogenesis; Culture; Differentiation; Marrow stromal stem cells; Microarray; Serum-free medium
Human stromal (mesenchymal) stem cells (hMSCs) are multipotent stem cells with ability to differentiate into mesoderm-type cells e.g. osteoblasts and adipocytes and thus they are being introduced into clinical trials for tissue regeneration. Traditionally, hMSCs have been isolated from bone marrow, but the number of cells obtained is limited. Here, we compared the MSC-like cell populations, obtained from alternative sources for MSC: adipose tissue and skin, with the standard phenotype of human bone marrow MSC (BM-MSCs). MSC from human adipose tissue (human adipose stromal cells (hATSCs)) and human skin (human adult skin stromal cells, (hASSCs) and human new-born skin stromal cells (hNSSCs)) grew readily in culture and the growth rate was highest in hNSSCs and lowest in hATSCs. Compared with phenotype of hBM-MSC, all cell populations were CD34−, CD45−, CD14−, CD31−, HLA-DR−, CD13+, CD29+, CD44+, CD73+, CD90+,and CD105+. When exposed to in vitro differentiation, hATSCs, hASSCs and hNSSCs exhibited quantitative differences in their ability to differentiate into adipocytes and to osteoblastic cells. Using a microarray-based approach we have unveiled a common MSC molecular signature composed of 33 CD markers including known MSC markers and several novel markers e.g. CD165, CD276, and CD82. However, significant differences in the molecular phenotype between these different stromal cell populations were observed suggesting ontological and functional differences. In conclusion, MSC populations obtained from different tissues exhibit significant differences in their proliferation, differentiation and molecular phenotype, which should be taken into consideration when planning their use in clinical protocols.
Electronic supplementary material
The online version of this article (doi:10.1007/s12015-012-9365-8) contains supplementary material, which is available to authorized users.
Stromal cells; Mesenchymal stem cell; Adipose tissue; Bone marrow; Skin; DNA microarray
Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.
The mesenchymal stroma harbors an important population of cells that possess stem cell-like characteristics including self renewal and differentiation capacities and can be derived from a variety of different sources. These multipotent mesenchymal stem cells (MSC) can be found in nearly all tissues and are mostly located in perivascular niches. MSC have migratory abilities and can secrete protective factors and act as a primary matrix for tissue regeneration during inflammation, tissue injuries and certain cancers.
These functions underlie the important physiological roles of MSC and underscore a significant potential for the clinical use of distinct populations from the various tissues. MSC derived from different adult (adipose tissue, peripheral blood, bone marrow) and neonatal tissues (particular parts of the placenta and umbilical cord) are therefore compared in this mini-review with respect to their cell biological properties, surface marker expression and proliferative capacities. In addition, several MSC functions including in vitro and in vivo differentiation capacities within a variety of lineages and immune-modulatory properties are highlighted. Differences in the extracellular milieu such as the presence of interacting neighbouring cell populations, exposure to proteases or a hypoxic microenvironment contribute to functional developments within MSC populations originating from different tissues, and intracellular conditions such as the expression levels of certain micro RNAs can additionally balance MSC function and fate.
We describe a simple method for bone engineering using biodegradable scaffolds with mesenchymal stem cells derived from human induced-pluripotent stem cells (hiPS-MSCs). The hiPS-MSCs expressed mesenchymal markers (CD90, CD73, and CD105), possessed multipotency characterized by tri-lineages differentiation: osteogenic, adipogenic, and chondrogenic, and lost pluripotency – as seen with the loss of markers OCT3/4 and TRA-1-81 – and tumorigenicity. However, these iPS-MSCs are still positive for marker NANOG. We further explored the osteogenic potential of the hiPS-MSCs in synthetic polymer polycaprolactone (PCL) scaffolds or PCL scaffolds functionalized with natural polymer hyaluronan and ceramic TCP (PHT) both in vitro and in vivo. Our results showed that these iPS-MSCs are functionally compatible with the two 3D scaffolds tested and formed typically calcified structure in the scaffolds. Overall, our results suggest the iPS-MSCs derived by this simple method retain fully osteogenic function and provide a new solution towards personalized orthopedic therapy in the future.
We previously demonstrated that human pericytes, which encircle capillaries and microvessels, give rise in culture to genuine mesenchymal stem cells (MSCs). This raised the question as to whether all MSC are derived from pericytes. Pericytes and other cells defined on differential expression of CD34, CD31, and CD146 were sorted from the stromal vascular fraction of human white adipose tissue. Besides pericytes, CD34+ CD31- CD146- CD45- cells, which reside in the outmost layer of blood vessels, the tunica adventitia, natively expressed MSC markers and gave rise in culture to clonogenic multipotent progenitors identical to standard bone marrow-derived MSC. Despite common MSC features and developmental properties, adventitial cells and pericytes retain distinct phenotypes and genotypes through culture. However, in the presence of growth factors involved in vascular remodeling, adventitial cells acquire a pericytes-like phenotype. In conclusion, we demonstrate the co-existence of 2 separate perivascular MSC progenitors: pericytes in capillaries and microvessels and adventitial cells around larger vessels.
Mesenchymal stromal cells (MSCs) originally isolated from bone marrow have multipotent differentiation potential, and favorable immunomodulatory and anti-inflammatory properties that make them very attractive for regenerative cellular therapy. Cells with similar phenotypic characteristics have now been derived from almost all fetal, neonatal and adult tissues; furthermore, more recently similar cells have also been generated from human embryonic stem cells (ESCs). Generation of MSCs from human ESCs provides an opportunity to study the developmental biology of human mesenchymal lineage generation in vitro. Generation of bone and cartilage from human ESC-derived MSCs and their functional characterization, both in vitro and in vivo, is also an active area of investigation as ESCs could provide an unlimited source of MSCs for potential repair of bone and cartilage defects. MSCs from adult sources are being investigated in numerous phase I-III clinical trials for a wide variety of indications, mainly based on their immunomodulatory properties. Our group and others have shown MSCs derived from human ESCs possess immunomodulatory properties similar to bone marrow-derived MSCs. Immunomodulatory properties of ESC-derived MSCs could prove to be highly valuable for their potential clinical applications in the future as derivatives of human ESCs have already entered clinical arena in the context of phase-I clinical trials.
Embryonic stem cells; mesenchymal stromal cells; mesenchymal stem cells
Multipotent mesenchymal stromal cells (MSCs) are found in a variety of adult tissues including human dermis. These MSCs are morphologically similar to bone marrow–derived MSCs, but are of unclear phenotype. To shed light on the characteristics of human dermal MSCs, this study was designed to identify and isolate dermal MSCs by a specific marker expression profile, and subsequently rate their mesenchymal differentiation potential. Immunohistochemical staining showed that MSC markers CD73/CD90/CD105, as well as CD271 and SSEA-4, are expressed on dermal cells in situ. Flow cytometric analysis revealed a phenotype similar to bone marrow–derived MSCs. Human dermal cells isolated by plastic adherence had a lower differentiation capacity as compared with bone marrow–derived MSCs. To distinguish dermal MSCs from differentiated fibroblasts, we immunoselected CD271+ and SSEA-4+ cells from adherent dermal cells and investigated their mesenchymal differentiation capacity. This revealed that cells with increased adipogenic, osteogenic, and chondrogenic potential were enriched in the dermal CD271+ population. The differentiation potential of dermal SSEA-4+ cells, in contrast, appeared to be limited to adipogenesis. These results indicate that specific cell populations with variable mesenchymal differentiation potential can be isolated from human dermis. Moreover, we identified three different subsets of dermal mesenchymal progenitor cells.
Adipose tissue represents an accessible, abundant, and replenishable source of adult stem cells for potential applications in regenerative medicine. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) resemble bone marrow-derived mesenchymal stem cells (BM-MSCs) with respect to morphology, immune-phenotype and multiple differentiation capability. In the present study, we investigated the feasibility of AT-MSC-based liver gene delivery for the treatment of alpha 1-antitrypsin deficiency.
Mouse AT-MSCs were tranduced by rAAV vectors and transplanted into mouse liver
We showed that AT-MSCs can be transduced by recombinant adeno-associated viral vector serotype 1 (rAAV1-CB-hAAT). After transplanting to mouse liver, ex vivo transduced AT-MSCs expressed the transgene product, human alpha 1-antitrypsin (hAAT). Importantly, serum levels of hAAT were sustained and no anti-hAAT antibody was detected in any recipients.
These results demonstrated that AT-MSCs can be transduced by rAAV vectors, engrafted into recipient livers, contribute to liver regeneration, and serve as a platform for transgene expression without eliciting an immune response. AT-MSC-based gene therapy presents a novel approach for the treatment of liver diseases, such as AAT deficiency.
Alpha 1 antitrypsin deficiency; Adeno-associated virus (AAV); Liver gene therapy; Liver regeneration
Multiple surface markers have been utilized for the enrichment of bone marrow mesenchymal stromal cells (MSCs) and to define primitive stem cells. We classified human bone marrow-derived MSC populations according to tissue nonspecific alkaline phosphatase (TNAP) activity. TNAP expression varied among unexpanded primary MSCs, and its level was not related to colony-forming activity or putative surface markers, such as CD105 and CD29, donor age, or gender. TNAP levels were increased in larger cells, and a colony-forming unit-fibroblast assay revealed that the colony size was decreased during in vitro expansion. TNAP-positive (TNAP+) MSCs showed limited multipotential capacity, whereas TNAP-negative (TNAP−) MSCs retained the differentiation potential into 3 lineages (osteogenic-, adipogenic-, and chondrogenic differentiation). High degree of calcium mineralization and high level of osteogenic-related gene expression (osteopontin, dlx5, and cbfa1) were found in TNAP+ cells. In contrast, during chondrogenic differentiation, type II collagen was successfully induced in TNAP− cells, but not in TNAP+ cells. TNAP+ cells showed high levels of the hypertrophic markers, type X collagen and cbfa1. Mesenchymal stem cell antigen-1 (MSCA-1) is identical to TNAP. Therefore, TNAP+ cells were sorted by using antibody targeting MSCA-1. MSCA-1-positive cells sorted for TNAP+ cells exhibited low proliferation rates. Expression of cell cycle-related genes (cyclin A2, CDK2, and CDK4) and pluripotency marker genes (rex1 and nanog) were higher in TNAP− MSC than in TNAP+ MSC. Therefore, TNAP− cells can be described as more primitive bone marrow-derived cells and TNAP levels in MSCs can be used to predict chondrocyte hypertrophy or osteogenic capacity.
Various source-derived mesenchymal stem cells (MSCs) have been considered for cell therapeutics in incurable diseases. To characterize MSCs from different sources, we compared human bone marrow (BM), adipose tissue (AT), and umbilical cord blood-derived MSCs (UCB-MSCs) for surface antigen expression, differentiation ability, proliferation capacity, clonality, tolerance for aging, and paracrine activity. Although MSCs from different tissues have similar levels of surface antigen expression, immunosuppressive activity, and differentiation ability, UCB-MSCs had the highest rate of cell proliferation and clonality, and significantly lower expression of p53, p21, and p16, well known markers of senescence. Since paracrine action is the main action of MSCs, we examined the anti-inflammatory activity of each MSC under lipopolysaccharide (LPS)-induced inflammation. Co-culture of UCB-MSCs with LPS-treated rat alveolar macrophage, reduced expression of inflammatory cytokines including interleukin-1α (IL-1α), IL-6, and IL-8 via angiopoietin-1 (Ang-1). Using recombinant Ang-1 as potential soluble paracrine factor or its small interference RNA (siRNA), we found that Ang-1 secretion was responsible for this beneficial effect in part by preventing inflammation. Our results demonstrate that primitive UCB-MSCs have biological advantages in comparison to adult sources, making UCB-MSCs a useful model for clinical applications of cell therapy.
umbilical cord blood; bone marrow; adipo tissue; mesenchymal stem cell; expansion; senescence; anti-inflammation; angiopoietin-1; cell therapy
Mesenchymal Stem cells (MSC) are now presented with the opportunities of multifunctional therapeutic approaches. Several reports are in support of their self-renewal, capacity for multipotent differentiation, and immunomodulatory properties. They are unique to contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose. In addition to promising trials in regenerative medicine, such as in the treatment of major bone defects and myocardial infarction, MSC has shown a therapeutic effect other than direct hematopoiesis support in hematopoietic reconstruction. MSCs are identified by the expression of many molecules including CD105 (SH2) and CD73(SH3/4) and are negative for the hematopoietic markers CD34, CD45, and CD14. Manufacturing of MSC for clinical trials is also an important aspect as their differentiation, homing and Immunomodulatory properties may differ. Their suppressive effects on immune cells, including T cells, B cells, NK cells and DC cells, suggest MSCs as a novel therapy for GVHD and other autoimmune disorders. Since the cells by themselves are non-immunogenic, tissue matching between MSC donor and recipient is not essential and, MSC may be the first cell type able to be used as an “off-the-shelf” therapeutic product. Following a successful transplantation, the migration of MSC to the site of injury refers to the involvement of chemokines and chemokine receptors of respective specificity. It has been demonstrated that cultured MSCs have the ability to engraft into healthy as well as injured tissue and can differentiate into several cell types in vivo, which facilitates MSC to be an ideal tool for regenerative therapy in different disease types. However, some observations have raised questions about the limitations for proper use of MSC considering some critical factors that warn regular clinical use.
Mesenchymal stem cell; MSC therapy; immunology; MSC application limitations
Mesenchymal stem cells (MSCs) are the most popular among the adult stem cells in tissue engineering and regenerative medicine. Since their discovery and functional characterization in the late sixties and early seventies, MSCs or MSC-like cells have been obtained from various mesodermal and non-mesodermal tissues, although majority of the therapeutic applications involved bone marrow derived MSCs. Based on its mesenchymal origin, it was predicted earlier that MSCs only can differentiate into mesengenic lineages like bone, cartilage, fat or muscle. However, varied isolation and cell culturing methods identified subsets of MSCs in the bone marrow which not only differentiated into mesenchymal lineages, but also into ectodermal and endodermal derivatives. Although, true pluripotent status is yet to be established, MSCs have been successfully used in bone and cartilage regeneration in osteoporotic fracture and arthritis respectively and in the repair of cardiac tissue following myocardial infarction. Immunosuppressive properties of MSCs extend utility of MSCs to reduce complications of graft versus host disease and rheumatoid arthritis. Homing of MSCs to sites of tissue injury, including tumor, is well established. In addition to their ability in tissue regeneration, MSCs can be genetically engineered ex vivo for delivery of therapeutic molecule(s) to the sites of injury or tumorigenesis as cell therapy vehicles. MSCs tend to lose surface receptors for trafficking and have been reported to develop sarcoma in long-term culture. In this article, we reviewed the current status of MSCs with special emphasis to therapeutic application in bone-related diseases.
Tissue regeneration and recovery in the adult body depends on self-renewal and differentiation of stem and progenitor cells. Mesenchymal stem cells (MSCs) that have the ability to differentiate into various cell types, have been isolated from the stromal fraction of virtually all tissues. However, little is known about the true identity of MSCs. MSC populations exhibit great tissue-, location- and patient-specific variation in gene expression and are heterogeneous in cell composition.
Our aim was to analyze the dynamics of differentiation of two closely related stromal cell types, adipose tissue-derived MSCs (AdMSCs) and dermal fibroblasts (FBs) along adipogenic, osteogenic and chondrogenic lineages using multiplex RNA-seq technology. We found that undifferentiated donor-matched AdMSCs and FBs are distinct populations that stay different upon differentiation into adipocytes, osteoblasts and chondrocytes. The changes in lineage-specific gene expression occur early in differentiation and persist over time in both AdMSCs and FBs. Further, AdMSCs and FBs exhibit similar dynamics of adipogenic and osteogenic differentiation but different dynamics of chondrogenic differentiation.
Our findings suggest that stromal stem cells including AdMSCs and dermal FBs exploit different molecular mechanisms of differentiation to reach a common cell fate. The early mechanisms of differentiation are lineage-specific and are similar for adipogenic and osteogenic differentiation but are distinct for chondrogenic differentiation between AdMSCs and FBs.