Extracellular calcium stimulates osteogenic differentiation of human adipose-derived stem cells by enhancing bone morphogenetic protein-2 expression

Bone morphogenetic protein-2 (BMP-2) promotes the differentiation of non-osteogenic mesenchymal cells to osteogenic cells. In this study, we isolated human adipose-derived stem cells (hASCs) and investigated the effects of recombinant human BMP-2 (rhBMP-2) and extracellular Ca²⁺ concentration ([Ca²⁺]_out) on the osteogenic differentiation of hASCs. rhBMP-2 promoted calcium deposition in hASCs and stimulated the mRNA expressions of six proteins known to be involved in the osteogenic differentiation of hASCs: Runx2, osterix, alkaline phosphatase, osteonectin, bone sialoprotein and osteocalcin. Elevation of [Ca²⁺]_out enhanced the level of alkaline phosphatase enzyme, increased the mRNA expressions of Runx2 and osteocalcin and induced the expressions of BMP-2 mRNA and protein in hASCs. Elevation of [Ca²⁺]_out transiently increased the intracellular Ca²⁺ concentration ([Ca²⁺]_in) due to activation of the calcium-sensing receptor (CaSR). The Ca²⁺-induced expressions of BMP-2 mRNA and protein were inhibited by the calmodulin antagonist, W-7. Furthermore, elevation of [Ca²⁺]_out decreased the cytoplasmic level of phosphorylated nuclear factor of activated T-cell-2 (NFAT-2) and increased the nuclear level of dephosphorylated NFAT2. Taken together, these results suggest that rhBMP-2 promotes the osteogenic differentiation of hASCs. Furthermore, an increase in [Ca²⁺]_out enhances the expression of BMP-2 via activation of the CaSR, elevation of [Ca²⁺]_in and stimulation of Ca²⁺/calmodulin-dependent NFAT-signaling pathways.     

Hypoxia enhances the viability,growth and chondrogenic potential of cryopreserved human adipose-derived stem cells

Cryopreservation is the only existing method of storage of human adipose-derived stem cells (ASCs) for clinical use. However, cryopreservation has been shown to be detrimental to ASCs, particularly in term of cell viability. To restore the viability of cryopreserved ASCs, it is proposed to culture the cells in a hypoxic condition. To this end, we aim to investigate the effect of hypoxia on the cryopreserved human ASCs in terms of not only cell viability, but also their growth and stemness properties, which have not been explored yet. In this study, human ASCs were cultured under four different conditions: fresh (non-cryopreserved) cells cultured in 1) normoxia (21% O₂) and 2) hypoxia (2% O₂) and cryopreserved cells cultured in 3) normoxia and 4) hypoxia. ASCs at passage 3 were subjected to assessment of viability, proliferation, differentiation, and expression of stemness markers and hypoxia-inducible factor-1 alpha (HIF-1α). We found that hypoxia enhances the viability and the proliferation rate of cryopreserved ASCs. Further, hypoxia upregulates HIF-1α in cryopreserved ASCs, which in turn activates chondrogenic genes to promote chondrogenic differentiation. In conclusion, hypoxic-preconditioned cryopreserved ASCs could be an ideal cell source for cartilage repair and regeneration.     

Development of a dual-wavelength fluorescent nanoprobe for in vivo and in vitro cell tracking consecutively

Many imaging probes have been developed for a wide variety of imaging modalities. However, no optical imaging probe could be utilized for both microscopic and whole animal imaging. To fill the gap, the dual-wavelength fluorescent imaging nanoprobe was developed to simultaneously carry both visible-range fluorescent dye and near-infrared (NIR) dye. Emission scan confirms that the nanoprobe exhibits two separate peaks with strong fluorescent intensity in both visible and NIR ranges. Furthermore, the dual-wavelength fluorescent nanoprobe has high photostability and colloidal stability, as well as long shelf-life. In vitro cell culture experiments show that the nanoprobe has the ability to label different types of cells (namely, esophageal, prostate, fibroblast and macrophage cell) for fluorescent microscope imaging. More importantly, cell tracking experiments confirm that cell migration and distribution in various organs can be tracked in real time using in vivo whole-body NIR imaging and in vitro microscopic imaging, respectively.     

Noninvasive in vivo cell tracking using molecular imaging: A useful tool for developing mesenchymal stem cell-based cancer treatment

Mounting evidence has emphasized the potential of cell therapies in treating various diseases by restoring damaged tissues or replacing defective cells in the body. Cell therapies have become a strong therapeutic modality by applying noninvasive in vivo molecular imaging for examining complex cellular processes, understanding pathophysiological mechanisms of diseases, and evaluating the kinetics/dynamics of cell therapies. In particular, mesenchymal stem cells (MSCs) have shown promise in recent years as drug carriers for cancer treatment. They can also be labeled with different probes and tracked in vivo to assess the in vivo effect of administered cells, and to optimize therapy. The exact role of MSCs in oncologic diseases is not clear as MSCs have been shown to be involved in tumor progression and inhibition, and the exact interactions between MSCs and specific cancer microenvironments are not clear. In this review, a multitude of labeling approaches, imaging modalities, and the merits/demerits of each strategy are outlined. In addition, specific examples of the use of MSCs and in vivo imaging in cancer therapy are provided. Finally, present limitations and future outlooks in terms of the translation of different imaging approaches in clinics are discussed.     

Pluripotency potential of human adipose-derived stem cells marked with exogenous green fluorescent protein

Musculoskeletal tissues regeneration requires rapid expansion of seeding cells both in vitro and in vivo while maintaining their multilineage differentiation ability. Human adipose-derived stem cells (ASCs) are considered to contain multipotent mesenchymal stem cells. Monolayer cultures of human ASCs were isolated from human lipoaspirates and passaged 3 times and then infected with replication-incompetent adenoviral vectors carrying green fluorescent protein (Ad/GFP) genes. Then, Ad/GFP infected human ASCs were transferred to osteogenic, chondrogenic, adipogenic, and myogenic medium. The morphological characterization of induced cells was observed using phase-contrast microscopy and fluorescence microscopy. The expression of marker proteins or genes was measured by immunocytochemical and RT-PCR analysis. Osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, aggrecan and SOX9 were positive in chondrogenic ones, peroxisome proliferator-activated receptor (PPAR-γ2) and lipoprotein lipase (LPL) were positive in adipogenic ones, and myogenin and myod1 was positive in myogenic ones. At the same time, the results of fluorescence microscopic imaging proved that the high level of GFP expression during ASCs differentiation maintained stable nearly 2 months. So the exogenous GFP and multilineage potential of human ASCs had no severe influences on each other. Since the human ASCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of GFP facilitates the research on ASCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.     

Co-culture with neurotrophic factor secreting cells induced from adipose-derived stem cells: Promotes neurogenic differentiation

Adipose-derived stem cells (ADSCs) and bone marrow stem cells (BMSCs) can be equally proper in the treatment of neurodegenerative diseases. However, ADSCs have practical benefits. In this study, we attempted to induce the secretion of neurotrophic factors (NTF) in human ADSCs. We then evaluated the effects of co-culture with NTF secreting cells in neural differentiation of human ADSCs. Isolated human ADSCs were induced to neurotrophic factors secreting cells. To evaluate the in vitro effects of NTF-secreting ADSCs on neurogenic differentiation of ADSCs, we used neurogenic induction medium (control group), the combination of neurogenic medium and conditioned medium, or co-cultured NTF-secreting ADSCs which were encapsulated in alginate beads (co-culture) for 7 days. ELISA showed increased (by about 5 times) release of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in NTF-secreting ADSCs compared to human ADSCs. Real time RT-PCR analysis revealed that NTF-secreting ADSCs highly expressed NGF and BDNF. In addition, co-culture with NTF-secreting ADSCs could also promote neuronal differentiation relative to gliogenesis. Overall, NTF-secreting ADSCs secrete a range of growth factors whose levels in culture could promote neuronal differentiation and could support the survival and regeneration in a variety of neurodegenerative diseases.     

Human adipose-derived mesenchymal stem cells: serial passaging,doubling time and cell senescence

Adult adipose-derived mesenchymal stem cells (AD-MSC) are very interesting to our research group because they are easy to harvest, they are abundant in humans, and they have potential clinical applications in autologous cell therapy for disc degeneration. We examined these cells through sequential serial passages to assess osteogenic and chondrogenic capabilities, mean doubling time and cell senescence. Osteogenic and chondrogenic potencies were maintained through 13 passages. Mean passage doubling time increased significantly with increasing passage number. When donor age was evaluated, passages 1-4 from older donors had significantly longer doubling times compared to cells from younger donors. Passages 5-11 showed similar findings when analyzed by donor age. The mean percent senescence increased significantly with cell passaging, rising from 0% at passage 1 to 3.4% at passage 13. These novel data suggest that caution should be exercised when using AD-MSC with long passage times.     

A rapid and efficient method for primary culture of human adipose-derived stem cells

Adipose tissue contains some populations, adipose-derived stem cells (ADSCs) which can differentiate into adipogenic, chondrogenic, osteogenic, myogenic, and endothelial cells. Furthermore, adipose tissue can be easily obtained in large quantities through a simple liposuction. ADSCs are thought to be an alternate source of autologous adult stem cells for cell-based therapy. However, it is time-consuming and inefficient to harvest ADSCs by using a traditional collagenase-digestion method. To meet the demand of large quantities of ADSCs in the basic and applied research of regenerative medicine, we developed a rapid and efficient method for isolation and culture of primary ADSCs. The results indicated that the ADSCs obtained with our method possessed strong abilities of proliferation and colony formation in vitro, and could keep low level of cell senescence with stable population doubling during long-term culture in vitro. Furthermore, these harvested ADSCs were capable to differentiate into osteogenic and adipogenic lineages in the specific induction medium. In addition, the results of flow cytometry analysis indicated that these ADSCs could positively express multiple CD markers, such as CD44, CD105, CD29, CD90, and CD13, and hardly expressed CD31, CD34, CD45, and CD106, which was homologous to the mesenchymal stem cells. Therefore, the ADSCs isolated with our method are consistent with previously reported characteristics of the ADSCs. This new method that we established in this study is an efficient tool to isolate and culture the stem cells from adipose tissue.     

The effects of adipose-derived stem cells on CD133-expressing bladder cancer cells

Mesenchymal stem cells (MSCs) hold great promise as therapeutic agents in regenerative medicine. They are also considered as a preferred cell source for urinary tract reconstruction. However, as MSCs exhibit affinity to tumor microenvironment, possible activation of tumor-initiating cells remains a major concern in the application of stem cell-based therapies for patients with a bladder cancer history. To analyze the influence of adipose-derived stem cells (ASCs) on bladder cancer cells with stem cell-like properties, we isolated CD133-positive bladder cancer cells and cultured them in conditioned medium from ASCs (ASC-CM). Our results showed that parental 5637 and HB-CLS-1 cells showed induced clonogenic potential when cultured in ASC-CM. Soluble mediators secreted by ASCs increased proliferation and viability of unsorted cells as well as CD133+ and CD133− subpopulations. Furthermore, incubation with ASC-CM modulated activation of intracellular signaling pathways. Soluble mediators secreted by ASCs increased phosphorylation of AKT1/2/3 (1.4-fold, P < 0.05), ERK1/2 (1.6-fold, P < 0.02), and p70 S6K (1.4-fold) in CD133+ cells isolated from 5637 cell line. In turn, decreased phosphorylation of those three proteins involved in PI3K/Akt and MAPK signaling was observed in CD133+ cells isolated from HB-CLS-1 cell line. Our results revealed that bladder cancer stem-like cells are responsive to signals from ASCs. Paracrine factors secreted by locally-delivered ASCs may, therefore, contribute to the modulation of signaling pathways involved in cancer progression, metastasis, and drug resistance.     

Differentiation of reprogrammed human adipose mesenchymal stem cells toward neural cells with defined transcription factors

Somatic cell reprogramming may become a powerful approach to generate specific human cell types for cell-fate determination studies and potential transplantation therapies of neurological diseases. Here we report a reprogramming methodology with which human adipose stem cells (hADSCs) can be differentiated into neural cells. After being reprogrammed with polycistronic plasmid carrying defined factor OCT3/4, SOX2, KLF4 and c-MYC, and further treated with neural induce medium, the hADSCs switched to differentiate toward neural cell lineages. The generated cells had normal karyotypes and exogenous vector sequences were not inserted in the genomes. Therefore, this cell lineage conversion methodology bypasses the risk of mutation and gene instability, and provides a novel strategy to obtain patient-specific neural cells for basic research and therapeutic application.     

Efficient generation of functional Schwann cells from adipose-derived stem cells in defined conditions

Schwann cells (SCs) are hitherto regarded as the most promising candidates for viable cell-based therapy to peripheral nervous system (PNS) injuries or degenerative diseases. However, the extreme drawbacks of transplanting autologous SCs for clinical applications still represent a significant bottleneck in neural regenerative medicine, mainly owing to the need of sacrificing a functional nerve to generate autologous SCs and the nature of slow expansion of the SCs. Thus, it is of great importance to establish an alternative cell system for the generation of sufficient SCs. Here, we demonstrated that adipose-derived stem cells (ADSCs) of rat robustly give rise to morphological, phenotypic and functional SCs using an optimized protocol. After undergoing a 3-week in vitro differentiation, almost all of treated ADSCs exhibited spindle shaped morphology similar to genuine SCs and expressed SC markers GFAP and S100. Most importantly, apart from acquisition of SC antigenic and biochemical features, the ADSC-derived SCs were functionally identical to native SCs as they possess a potential ability to form myelin, and secret nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF). The current study may provide an ideal strategy for harvesting sufficient SCs for cell-based treatment of various peripheral nerve injuries or disorders.     

Effects of human adipose-derived stem cells on the viability of rabbit random pattern flaps

Background aimsFlap necrosis is the most commonly encountered outcome influencing the effect of operations in clinical practice. The advent of cytotherapy and regenerative medicine with stem cells, especially adipose-derived stem cell therapy, appears to be a promising approach in providing multi-lineage differentiating cells. However, autologous stem cells are limited in both quantity and quality in aging individuals. Hence, xenogenic stem cell therapy was used in this study.MethodsRandom pattern flaps (6 cm × 2 cm) were prepared in a rabbit model transplanted either with 4 × 10⁵ human adipose-derived stem cells at five sites or equal volumes of Dulbecco's modified Eagle's medium. At 7 days after operation, the viability of the flaps from both groups was evaluated. We determined the numbers of locally infiltrating T cells, whereas the CD4/CD8 ratio, interferon, interleukin (IL)-2, IL-4 and IL-10 in the serum were determined to evaluate the immunological response of the rabbit. Moreover, Dil labeling was administrated to trace the homing of the transplanted stem cells.ResultsBoth the survival areas and the capillary number of the flaps that were injected with human adipose-derived stem cells significantly increased as compared with the control group (P < 0.05). Additionally, no significant difference in the immune response was detected between the groups. Dil-labeled stem cells were found to participate in the formation of tubular structures, which were further shown to be CD31+, although not predominantly.ConclusionsHuman adipose-derived stem cells could be used therapeutically to improve the viability of random pattern flaps without detection of serious immune rejection of stem cells.     

Defined serum-free media for in vitro expansion of adipose-derived mesenchymal stem cells

BackgroundThere is a growing interest in mesenchymal stem cells (MSCs) because they are regarded as good candidates for cell therapy. Adipose tissue represents an easily accessible source to derive mesenchymal stem cells (Ad-MSCs) non-invasively in large numbers. The aim of this study was to evaluate a defined serum-free medium for in vitro expansion of MSCs as a prerequisite for their clinical use.MethodsAdipose tissue was isolated from healthy donors. Cells were isolated and expanded for five passages in serum-free medium (Mesencult-XF) and Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (DMEM-FBS). MSC morphology, marker expression, viability, population doubling time and differentiation potential toward osteogenic and adipogenic lineages were evaluated. Bone marrow MSCs were included as controls.ResultsAd-MSCs cultured in Mesencult-XF had shorter population doubling time (33.3 ± 13.7 h) compared with those cultured in DMEM-FBS (54.3 ± 41.0 h, P < 0.05). Ad-MSCs cultured in Mesencult-XF displayed a stable morphology and surface marker expression and a higher differentiation potential in comparison to Ad-MSCs cultured in DMEM-FBS.ConclusionsThe defined serum-free and xeno-free Mesencult-XF media appear to be a good choice for Ad-MSCs, but it is not as good in supporting culture of bone marrow MSCs when the cells are to be used for clinical purposes.     

Biological character of human adipose-derived adult stem cells and influence of donor age on cell replication in culture

To investigate the biological character of human adipose-derived adult stem cells (hADAS cells) when cultured in vitro and the relationship between hADAS cell's replication activity and the donor's age factor, and to assess the stem cells as a new source for tissue engineering, hADAS cells are isolated from human adipose tissue of different age groups (from adolescents to olds ＜20 years old, 21-40years old, 41-60 years old and ＞61 years old groups). The protein markers (CD29, CD34, CD44, CD45,CD49d, HLA-DR, CD106) of hADAS cells were detected by flow cytometry (FCM) to identify the stem cell,and the cell cycle was examined for P20 hADAS cells to evaluate the safety of the subculture in vitro.The generative activity of hADAS cells in different age groups was also examined by MTT method. The formula "TD = t log2/logNt - logN0 "was used to get the time doubling (TD) of the cells. The results showed that the cells kept heredity stabilization by chromosome analysis for at least 20 passages. The TD of these cells increased progressively by ageing, and the TD of the ＜20 years old group was lower than that of the ＞61 years old group (statistical analysis of variance (ANOVA), P=-0.002, P＜0.05). These findings suggested that a higher level of hADAS cells replication activity was found in the younger donators, and they represent novel and valuable seed cells for studies of tissue engineering.     

Biological character of human adipose-derived adult stem cells and influence of donor age on cell replication in culture

To investigate the biological character of human adipose-derived adult stem cells (hADAS cells) when cultured in vitro and the relationship between hADAS cell’s replication activity and the donor’s age factor, and to assess the stem cells as a new source for tissue engineering. hADAS cells are isolated from human adipose tissue of different age groups (from adolescents to olds: <20 years old, 21–40 years old, 41–60 years old and >61 years old groups). The protein markers (CD29, CD34, CD44, CD45, CD49d, HLA-DR, CD106) of hADAS cells were detected by flow cytometry (FCM) to identify the stem cell, and the cell cycle was examined for P20 hADAS cells to evaluate the safety of the subculture in vitro. The generative activity of hADAS cells in different age groups was also examined by MTT method. The formula “TD = t log2/logNt − logN0” was used to get the time doubling (TD) of the cells. The results showed that the cells kept heredity stabilization by chromosome analysis for at least 20 passages. The TD of these cells increased progressively by ageing, and the TD of the <20 years old group was lower than that of the >61 years old group (statistical analysis of variance (ANOVA), P=0.002, P<0.05). These findings suggested that a higher level of hADAS cells replication activity was found in the younger donators, and they represent novel and valuable seed cells for studies of tissue engineering.