Human adipose tissue is a source of multipotent stem cells

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.     

Human breast milk is a rich source of multipotent mesenchymal stem cells

Putative stem cells have been isolated from various tissue fluids such as synovial fluid, amniotic fluid, menstrual blood, etc. Recently the presence of nestin positive putative mammary stem cells has been reported in human breast milk. However, it is not clear whether they demonstrate multipotent nature. Since human breast milk is a non-invasive source of mammary stem cells, we were interested in examining the nature of these stem cells. In this pursuit, we could succeed in isolating and expanding a mesenchymal stem cell-like population from human breast milk. These cultured cells were examined by immunofluorescent labeling and found positive for mesenchymal stem cell surface markers CD44, CD29, SCA-1 and negative for CD33, CD34, CD45, CD73 confirming their identity as mesenchymal stem cells. Cytoskeletal protein marker analysis revealed that these cells expressed mesenchymal stem cells markers, namely, nestin, vimentin, smooth muscle actin and also manifests presence of E-Cadherin, an epithelial to mesenchymal transition marker in their early passages. Further we tested the multipotent differentiation potential of these cells and found that they can differentiate into adipogenic, chondrogenic and oesteogenic lineage under the influence of specific differentiation cocktails. This means that these mesenchymal stem cells isolated from human breast milk could potentially be “reprogrammed” to form many types of human tissues. The presence of multipotent stem cells in human milk suggests that breast milk could be an alternative source of stem cells for autologous stem cell therapy although the significance of these cells needs to be determined.     

Adipose tissue as a potential source of hematopoietic stem/progenitor cells

It has been more than 30 years since adipose tissue (AT) has been recognized as a central modulator orchestrating sophisticated process termed "immunometabolism". Nonetheless, despite its unique involvement in the regulation of immune and endocrine homeostasis, recent studies demonstrated that AT also contains significant number of hematopoietic stem/progenitor cells (HSPCs) that may be there "settling down" throughout life. In this article we will focus on presenting the current concepts regarding endocrine, immunological, and molecular mechanisms that may contribute to and regulate bone marrow (BM)-derived HSPCs homing into AT environment, as well as, highlight various structural and morphological similarities between BM and AT that might be involved in creating appropriate tissue niches for BM-derived HSPCs in AT. Finally, we will discuss how development of obesity or type 2 diabetes may influence balance of homing signals for HSPCs in AT environment.     

Human marrow-derived mesenchymal progenitor cells

A number of adult mesenchymal tissues contain subpopulations of undifferentiated cells, which retain the capacity to differentiate along multiple lineages. These mesenchymal progenitor cells may be cultured in an undifferentiated state and, when given the appropriate signals, differentiate into an expanding list of several mesenchymal and a few ectodermal derived tissues. The maintenance and propagation of the multipotential nature of these progenitor cell populations are crucially dependent on the isolation protocol, the culture expansion conditions, particularly the properties of the fetal bovine serum supplement in the culture medium. This article describes a method for selection of the appropriate serum lot, and introduces a simplified isolation technique to optimize the yield of progenitor cells that maintain the capability of undergoing multilineage differentiation in response to appropriate cues. Cell populations isolated and culture expanded in this manner, by virtue of their multiple differentiation potential, should serve as ideal candidate cells for tissue engineering applications for the repair and regeneration of tissue damaged by disease and or trauma.     

Human subcutaneous adipose tissue subjected to cold shock as a source of viable cellular population with characteristics of multipotent mesenchymal stromal cells

Cellular population with characteristics of multipotent mesenchymal stromal cells (MMSCs) was isolated from subcutaneous adipose tissue frozen without any cryoprotectant at -70 degrees C. Under critical for the adipose tissue condition, the cells retained their viability in vitro and ability of adhesion to plastic. Cellular population was homogeneous and represented by small cells (d - 7 microm) with fibroblast-like morphology. Cells were positively stained with Abs for the Abs: CD29, CD44, CD49a, b, d, CD73, CD90, CD105, CD166, HLA ABC. Cells were negative for CD34, CD45--markers of hematopoietic cells, CD31--marker of endothelial cells, Stro-1, as well as for HLA DR, DP, DQ (flow cytometer analysis). Being induced to differentiate in vitro, the cells were able to differentiate into cells similar to cells of bone, adipose and cartilage tissue. Karyological assay of the cells isolated from human adipose tissue subjected to cold shock revealed diploid set of chromosomes, 46, XX, without aneuploidy and structural reconstructions of chromosomes. Thus, it has been established that, under extreme condition for the organism, the population of cells with a phenotype similar to miltipotent mesenchymal stromal cells is preserved in subcutaneous adipose tissue.     

Human mesenchymal and murine stromal cells support human lympho-myeloid progenitor expansion but not maintenance of multipotent haematopoietic stem and progenitor cells

A major goal in haematopoietic stem cell (HSC) research is to define conditions for the expansion of HSCs or multipotent progenitor cells (MPPs). Since human HSCs/MPPs cannot be isolated, NOD/SCID repopulating cell (SRC) assays emerged as the standard for the quantification of very primitive haematopoietic cell. However, in addition to HSCs/MPPs, lympho-myeloid primed progenitors (LMPPs) were recently found to contain SRC activities, challenging this assay as clear HSC/MPP readout. Because our revised model of human haematopoiesis predicts that HSCs/MPPs can be identified as CD133⁺CD34⁺ cells containing erythroid potentials, we investigated the potential of human mesenchymal and conventional murine stromal cells to support expansion of HSCs/MPPs. Even though all stromal cells supported expansion of CD133⁺CD34⁺ progenitors with long-term myeloid and long-term lymphoid potentials, erythroid potentials were exclusively found within erythro-myeloid CD133^lowCD34⁺ cell fractions. Thus, our data demonstrate that against the prevailing assumption co-cultures on human mesenchymal and murine stromal cells neither promote expansion nor maintenance of HSCs and MPPs.     

Human subcutaneous adipose tissue subjected to cold shock as source of viable cell population with characteristics of multipotent mesenchymal stromal cells

A cell population with characteristics of multipotent mesenchymal stromal cells (MMSCs) was isolated from human subcutaneous adipose tissue (SAT) submitted to a deep freezing (at −70°C) without cryoprotector. Under critical conditions for tissue, the cells retained their viability in vitro and were adhesive to plastic. The cell population was characterized by homogeneity and was represented by small cells (7 μm in diameter) with fibroblast-like morphology. The cytofluorimetric analysis of the cells revealed the presence of antigens on their surface whose expression is characteristic of MMSCs, including CD29, CD44, CD49a,b,d, CD73, CD90, CD105, CD166, and HLA ABC. The cells were negative for CD34, CD45, which are markers of hematopoietic cells; CD31, a marker of endothelial cells; and Stro-1, as well as antigens of the major histocompatibility complex of the II class HLA DR, DP, and DQ. On average, the proportion of cells that carry the receptor of the stem-cell factor c-kit (CD117) amounted to 3%. Upon induction to differentiation in vitro, these cells turned out to be able to form cells similar to cells of bone, adipose, and cartilage tissues. Karyological analysis (GTG staining method) demonstrated the diploid set of chromosomes of cells, without aneuploidy and structural reconstruction of chromosomes. Thus, it has been established that, in SAT submitted to low-temperature shock, a viable population of cells with a phenotype similar MMSCs is preserved.     

The human adipose tissue is a source of multipotent stem cells

Multipotent stem cells constitute an unlimited source of differentiated cells that could be used in pharmacological studies and in medicine. Recently, several publications have reported that adipose tissue contains a population of cells able to differentiate into different cell types including adipocytes, osteoblasts, myoblasts, and chondroblasts. More recently, stem cells with a multi-lineage potential at the single cell level have been isolated from human adipose tissue. These cells, called human Multipotent Adipose-Derived Stem (hMADS) cells, have been established in culture and interestingly, maintain their characteristics with long-term passaging. The adipocyte differentiation of hMADS cells has been thoroughly studied and differentiated cells exhibit the unique feature of human adipocytes. Finally, potential applications of stem cells isolated from adipose tissue in medicine will be discussed.     

Differentiation and regeneration potential of mesenchymal progenitor cells derived from traumatized muscle tissue

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.     

Cochlear epithelial of dog fetuses: a new source of multipotent stem cells

Hearing loss caused by the damage of cochlea sensory cells or neurons is a common human disease, but also affects dogs and other animals. To test their progenitor nature as potential value for future therapies, we characterized cells derived from the cochlear epithelium in dog fetuses. In total, 8 fetuses of 35–40 days of gestation, derived from castration campaigns, were investigated. Cells were analysed by the MTT colorimetric assay and in regard to cell cycle, differentiation capacities, immunophenotypes and qPCR analysis. In culture, cells had a fibroblast-like morphology. Phenotypic immunocharacterization showed positive staining for mesenchymal stem cell and pluripotency markers and were negative for hematopoietic cell markers. Cells possessed differentiation capacity for the three main cell lineages: osteogenic, adipogenic and chondrogenic, altogether indicating their nature as mesenchymal stem cells. Thus, cells derived from fetal cochlear tissues indeed may provide valuable sources of progenitor cells for cell therapy of canine deafness and other diseases.     

Generation and expansion of multipotent mesenchymal progenitor cells from cultured human pancreatic islets

Cellular models and culture conditions for in vitro expansion of insulin-producing cells represent a key element to develop cell therapy for diabetes. Initial evidence that human beta-cells could be expanded after undergoing a reversible epithelial-mesenchymal transition has been recently negated by genetic lineage tracing studies in mice. Here, we report that culturing human pancreatic islets in the presence of serum resulted in the emergence of a population of nestin-positive cells. These proliferating cells were mainly C-peptide negative, although in the first week in culture, proliferating cells, insulin promoter factor-1 (Ipf-1) positive, were observed. Later passages of islet-derived cells were Ipf-1 negative and displayed a mesenchymal phenotype. These human pancreatic islet-derived mesenchymal (hPIDM) cells were expanded up to 10(14) cells and were able to differentiate toward adipocytes, osteocytes and chondrocytes, similarly to mesenchymal stem/precursor cells. Interestingly, however, under serum-free conditions, hPIDM cells lost the mesenchymal phenotype, formed islet-like clusters (ILCs) and were able to produce and secrete insulin. These data suggest that, although these cells are likely to result from preexisting mesenchymal cells rather than beta-cells, hPIDM cells represent a valuable model for further developments toward future replacement therapy in diabetes.     

Human amnion-derived mesenchymal stem cells are a potential source for uterine stem cell therapy

Abstract. Objectives: Human amnion is an easy‐to‐obtain novel source of human mesenchymal stem cells, which poses little or no ethical dilemmas. We have previously shown that human amnion‐derived mesenchymal (HAM) cells exhibit certain mesenchymal stem cell‐like characteristics with respect to expression of stem cell markers and differentiation potentials. Materials and methods: In this study, we further characterized HAM cells’ potential for in vivo therapeutic application. Results: Flow cytometric analyses of HAM cells show that they express several stem cell‐related cell surface markers, including CD90, CD105, CD59, CD49d, CD44 and HLA‐ABC, but not CD45, CD34, CD31, CD106 or HLA‐DR. HAM cells at the 10th passage showed normal karyotype. More interestingly, the AbdB‐like HOXA genes HOXA9, HOXA10 and HOXA11 that are expressed in the mesenchyme of the developing female reproductive tract and pregnant uteri are also expressed in HAM cells, suggesting similarities between these two mesenchymal cell types. Progesterone receptor is also highly expressed in HAM cells and expression of genes or proteins in HAM cells could be manipulated with the aid of lentivirus technology or cell‐permeable peptides. To test potentials of HAM cells for in vivo application, we introduced enhanced green fluorescence protein (EGFP)‐expressing HAM cells to mice by intrauterine infusion (into uteri) or by intravenous injection (into the circulation). Presence of EGFP‐expressing cells within the uterine mesenchyme after intrauterine infusion or in lungs after intravenous injection was noted within 1–4 weeks. Conclusions: Collectively, these results suggest that HAM cells are a potential source of mesenchymal stem cells with therapeutic potential.     

Fat pad-derived mesenchymal stem cells as a potential source for cell-based adipose tissue repair strategies

Background: Mesenchymal stem cells are able to undergo adipogenic differentiation and present a possible alternative cell source for regeneration and replacement of adipose tissue. The human infrapatellar fat pad is a promising source of mesenchymal stem cells with many source advantages over from bone marrow. It is important to determine whether a potential mesenchymal stem‐cell exhibits tri‐lineage differentiation potential and is able to maintain its proliferation potential and cell‐surface characterization on expansion in tissue culture. We have previously shown that mesenchymal stem cells derived from the fat pad can undergo chondrogenic and osteogenic differentiation, and we characterized these cells at early passage. In the study described here, proliferation potential and characterization of fat pad‐derived mesenchymal stem cells were assessed at higher passages, and cells were allowed to undergo adipogenic differentiation. Materials and methods: Infrapatellar fat pad tissue was obtained from six patients undergoing total knee replacement. Cells isolated were expanded to passage 18 and proliferation rates were measured. Passage 10 and 18 cells were characterized for cell‐surface epitopes using a range of markers. Passage 2 cells were allowed to undergo differentiation in adipogenic medium. Results: The cells maintained their population doubling rates up to passage 18. Cells at passage 10 and passage 18 had cell‐surface epitope expression similar to other mesenchymal stem cells previously described. By staining it was revealed that they highly expressed CD13, CD29, CD44, CD90 and CD105, and did not express CD34 or CD56, they were also negative for LNGFR and STRO1. 3G5 positive cells were noted in cells from both passages. These fat pad‐derived cells had adipogenic differentiation when assessed using gene expression for peroxisome proliferator‐activated receptor γ2 and lipoprotein lipase, and oil red O staining. Discussion: These results indicate that the cells maintained their proliferation rate, and continued expressing mesenchymal stem‐cell markers and pericyte marker 3G5 at late passages. These results also show that the cells were capable of adipogenic differentiation and thus could be a promising source for regeneration and replacement of adipose tissue in reconstructive surgery.     

Invited review: mesenchymal progenitor cells in intramuscular connective tissue development

The abundance and cross-linking of intramuscular connective tissue contributes to the background toughness of meat, and is thus undesirable. Connective tissue is mainly synthesized by intramuscular fibroblasts. Myocytes, adipocytes and fibroblasts are derived from a common pool of progenitor cells during the early embryonic development. It appears that multipotent mesenchymal stem cells first diverge into either myogenic or non-myogenic lineages; non-myogenic mesenchymal progenitors then develop into the stromal-vascular fraction of skeletal muscle wherein adipocytes, fibroblasts and derived mesenchymal progenitors reside. Because non-myogenic mesenchymal progenitors mainly undergo adipogenic or fibrogenic differentiation during muscle development, strengthening progenitor proliferation enhances the potential for both intramuscular adipogenesis and fibrogenesis, leading to the elevation of both marbling and connective tissue content in the resulting meat product. Furthermore, given the bipotent developmental potential of progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which likely results in the overall improvement of marbling (more intramuscular adipocytes) and tenderness (less connective tissue) of meat. Fibrogenesis is mainly regulated by the transforming growth factor (TGF) β signaling pathway and its regulatory cascade. In addition, extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment for regulating myogenic differentiation of satellite cells and muscle growth. Despite rapid progress, many questions remain in the role of extracellular matrix on muscle development, and factors determining the early differentiation of myogenic, adipogenic and fibrogenic cells, which warrant further studies.     

Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells

Human liposuction aspirates contain pluripotent adipose-derived mesodermal stem cells that have previously been shown to differentiate into various mesodermal cell types, including osteoblasts and chondrocytes. To develop an autologous research model of bone and cartilage tissue engineering, the authors sought to determine whether rat inguinal fat pads contain a similar population of osteochondrogenic precursor cells. It was hypothesized that the rat inguinal fat pad contains adipose-derived multipotential cells that resemble human adipose-derived mesodermal stem cells in their osteochondrogenic capacity. To test this, the authors assessed the ability of cells isolated from the rat inguinal fat pad to differentiate into osteoblasts and chondrocytes by a variety of lineage-specific histologic stains.Rat inguinal fat pads were isolated and processed from Sprague-Dawley rats into a fibroblast-like cell population. Cell cultures were placed in pro-osteogenic media containing dexamethasone, ascorbic acid, and beta-glycerol phosphate. Osteogenic differentiation was assessed at 2, 4, and 6 weeks. Alkaline phosphatase activity and von Kossa staining were performed to assess osteoblastic differentiation and the production of a calcified extracellular matrix. Cell cultures were also placed in prochondrogenic conditions and media supplemented with transforming growth factor-beta1, insulin, transferrin, and ascorbic acid. Chondrogenic differentiation was assessed at 2, 7, and 14 days by the presence of positive Alcian blue staining and type II collagen immunohistochemistry. Cells placed in osteogenic conditions changed in structure to a more cuboidal shape, formed bone nodules, stained positively for alkaline phosphatase activity, and secreted calcified extracellular matrix by 2 weeks. Cells placed in chondrogenic conditions formed cartilaginous nodules within 48 hours that stained positively for Alcian blue and type II collagen. The authors identified the rat inguinal fat pad as a source of osteochondrogenic precursors and developed a straightforward technique to isolate osteochondrogenic precursors from a small animal source. This relatively easily obtained source of osteochondrogenic cells from the rat may be useful for study of tissue engineering strategies and the basic science of stem cell biology.     

The potential of adipose-derived adult stem cells as a source of neuronal progenitor cells

Adipose-derived adult stem cells are a population of mesenchymal stem cells extracted from discarded adipose tissue. Many have reported the differentiation of adipose-derived stem cells into chondrocytes, myocytes, osteoblasts, and, most recently, neural progenitor cells. This article covers the current state of the potential of the differentiation of adipose-derived stem cells into neuronal cells and an overview of their potential as adult stem therapies for neurological disorders. It has been reported that adipose-derived stem cells are capable of undergoing neuronal differentiation using protocols similar to that of Woodbury et al., which reported the differentiation of bone marrow stromal cells specifically into neurons. However, the transdifferentiation of bone marrow stromal cells into neuronal cells has recently fallen under intense criticism, which will likely place the plasticity of adipose-derived stem cells under scrutiny as well. To date, no group has produced evidence that adipose-derived stem cells are capable of differentiating to mature, functional neuronal cells in vitro. However, recent in vivo studies with adipose-derived stem cells are promising.