Craniofacial microsomia is a congenital deformity caused by asymmetric development of the skull (cranium) and face before birth. Current treatments include corrective surgery and replacement of the deformed structure using autograft tissue, which results in donor site morbidity. An alternative therapy can be achieved by developing a resorbable scaffold for skeletal muscle regeneration which will help restore the symmetry and function of the facial muscles and reduce donor site morbidity. Two resorbable weft knitted scaffolds were fabricated using poly(ε-caprolactone) multifilament yarns with unique auxetic design structures possessing negative Poisson’s ratio (NPR). These scaffolds exhibit their NPR elasticity through an increase in total volume as well as no lateral narrowing when stretched longitudinally, which can provide orientated mechanical supports to the cell growth of skeletal muscle regeneration. These scaffolds were evaluated for the required physical properties, mechanical performance and biocompatibility by culturing them with neonatal human dermal fibroblasts so as to determine their cell metabolic activity, cell attachment and proliferation. This study can facilitate the understanding and engineering of textile-based scaffolds for tissues/organs. The work also paves a pathway to emerge the NPR textiles into tissue engineering, which has an extensive potential for biomedical end-uses.

At the present time, there is no successful synthetic, off-the-shelf small-caliber vascular graft (<6 mm) for the repair or bypass of the coronary or carotid arteries. This stimulates on-going investigations to fabricate an artificial vascular graft that has both sufficient mechanical properties as well as superior biological performance. Collagen has long been considered as a viable material to encourage cell recruitment, tissue regeneration, and revascularization, but its use has been limited by its inferior mechanical properties. In this study, novel electrochemically aligned collagen filaments were used to engineer a bilayer small-caliber vascular graft, by circular knitting the collagen filaments and electrospinning collagen nanofibers. The collagen prototype grafts showed significantly greater bursting strength under dry and hydrated conditions to that of autografts such as the human internal mammary artery and the saphenous vein (SV). The suture retention strength was sufficient under dry condition, but that under hydrated condition needs to be further improved. The radial dynamic compliance of the collagen grafts was similar to that of the human SV. During in vitro cell culture assays with human umbilical vein endothelial cells, the prototype collagen grafts also encouraged cell adhesion and promoted cell proliferation compared to the synthetic poly(lactic acid) grafts. In conclusion, this study demonstrated the feasibility of the use of novel collagen filaments for fabricating small caliber tissue-engineered vascular grafts that provide both sufficient mechanical properties and superior biological performance.

Non-motile primary cilia are dynamic cellular sensory structures and are expressed in adipose-derived stem cells (ASCs). We have previously shown that primary cilia are involved in chemically-induced osteogenic differentiation of human ASC (hASCs) in vitro. Further, we have reported that 10% cyclic tensile strain (1 Hz, 4 hours/day) enhances hASC osteogenesis. We hypothesize that primary cilia respond to cyclic tensile strain in a lineage dependent manner and that their mechanosensitivity may regulate the dynamics of signaling pathways localized to the cilium. We found that hASC morphology, cilia length and cilia conformation varied in response to culture in complete growth, osteogenic differentiation, or adipogenic differentiation medium, with the longest cilia expressed in adipogenically differentiating cells. Further, we show that cyclic tensile strain both enhances osteogenic differentiation of hASCs while it suppresses adipogenic differentiation as evidenced by upregulation of RUNX2 gene expression and downregulation of PPARG and IGF-1, respectively. This study demonstrates that hASC primary cilia exhibit mechanosensitivity to cyclic tensile strain and lineage-dependent expression, which may in part regulate signaling pathways localized to the primary cilium during the differentiation process. We highlight the importance of the primary cilium structure in mechanosensing and lineage specification and surmise that this structure may be a novel target in manipulating hASC for in tissue engineering applications.

In this study, we report for the first time that the primary ciliumacts as a crucial sensor for electrical field stimulation (EFS)-enhanced osteogenic response in osteoprogenitor cells. In addition, primary cilia seem to functionally modulate effects of EFS-induced cellular calcium oscillations. Primary cilia are organelles that have recently been implicated to play a crucial sensor role for many mechanical and chemical stimuli on stem cells. Here, we investigate the role of primary cilia in EFS-enhanced osteogenic response of human adipose-derived stem cells (hASCs) by knocking down 2 primary cilia structural proteins, polycystin-1 and intraflagellar protein-88. Our results indicate that structurally integrated primary cilia are required for detection of electrical field signals in hASCs. Furthermore, by measuring changes of cytoplasmic calcium concentration in hASCs during EFS, our findings also suggest that primary cilia may potentially function as a crucial calcium-signaling nexus in hASCs during EFS.-Cai, S., Bodle, J. C., Mathieu, P. S., Amos, A., Hamouda, M., Bernacki, S., McCarty, G., Loboa, E. G. Primary cilia are sensors of electrical field stimulation to induce osteogenesis of human adipose-derived stem cells.

Scaffolds play an important role in tissue engineering by providing structural framework and a surface for cells to attach, proliferate, and secrete extracellular matrix (ECM). In order to enable efficient tissue formation, delivering sufficient cells into the scaffold three-dimensional (3D) matrix using traditional static and dynamic seeding methods continues to be a critical challenge. In this study, we investigate a new cell delivery approach utilizing deposition of hydrogel-cell encapsulated microspheroids into polycaprolactone (PCL) scaffolds to improve the seeding efficiency. Three-dimensional-bioplotted PCL constructs (0 deg/90 deg lay down, 284 ± 6 μm strand width, and 555 ± 8 μm strand separation) inoculated with MG-63 model bone cells encapsulated within electrostatically generated calcium-alginate microspheroids (Ø 405 ± 13 μm) were evaluated over seven days in static culture. The microspheroids were observed to be uniformly distributed throughout the PCL scaffold cross section. Encapsulated cells remained viable within the constructs over the test interval with the highest proliferation noted at day 4. This study demonstrates the feasibility of the new approach and highlights the role and critical challenges to be addressed to successfully utilize 3D-bioprinting for microencapsulated cell delivery.

Transdermal osseointegrated prostheses (TOPs) are emerging as an alternative to socket prostheses. Electron beam melting (EBM) is a promising additive manufacturing technology for manufacture of custom, freeform titanium alloy (Ti6Al4V) implants. Skin ongrowth for infection resistance and mechanical stability are critically important to the success of TOP, which can be influenced by material composition and surface characteristics. We assessed viability and proliferation of normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF) on several Ti6Al4V surfaces: solid polished commercial, solid polished EBM, solid unpolished EBM and porous unpolished EBM. Cell proliferation was evaluated at days 2 and 7 using alamarBlue(®) and cell viability was analyzed with a fluorescence-based live-dead assay after 1 week. NHDF and NHEK were viable and proliferated on all Ti6Al4V surfaces. NHDF proliferation was highest on commercial and EBM polished surfaces. NHEK was highest on commercial polished surfaces. All EBM Ti6Al4V discs exhibited an acceptable biocompatibility profile compared to solid Ti6Al4V discs from a commercial source for dermal and epidermal cells. EBM may be considered as an option for fabrication of custom transdermal implants.

Human adipose-derived stem cells (hASC) are now a prevalent source of adult stem cells for studies in tissue engineering and regenerative medicine. However, researchers utilizing hASC in their investigations often encounter high levels of donor-to-donor variability in hASC differentiation potential. Because of this, conducting studies with this primary cell type can require extensive resources to generate statistically significant data. We present a method to generate pooled donor cell populations, termed "superlots," containing cell populations derived from four to five age-clustered donors. The goal of generating these superlots was to 1) increase experimental throughput, 2) to utilize assay resources more efficiently, and 3) to begin to establish global hASC differentiation behaviors that may be associated with donor age. With our superlot approach, we have validated that pooled donor cell populations exhibit proliferative activity representing the combined behavior of each individual donor cell line. Further, the superlots also exhibit differentiation levels roughly approximating the average combined differentiation levels of each individual donor cell line. We established that high donor-to-donor variability exists between the pre-, peri-, and postmenopausal age groupings and that proliferation and differentiation characteristics can vary widely, independent of age. Interestingly, we did observe that cell lines derived from postmenopausal donors demonstrated a relatively high proclivity for osteogenic differentiation and a relatively lowered proclivity for adipogenic differentiation as compared with cells derived from pre- and perimenopausal donors. In general, superlots effectively represented the average differentiation behavior of each of their contributing cell populations and could provide a powerful tool for increasing experimental throughput to more efficiently utilize resources when studying hASC differentiation.

The material surface plays an important role in the case of biomaterials used as tissue engineering scaffolds. On exposure to a biological environment, extra cellular matrix (ECM) proteins are adsorbed non-specifically onto the surface and cells interact indirectly with the surface through the adsorbed proteins. Most synthetic polymeric biomaterials lack the desirable surface properties for cells as well as have poor cellular adhesion due to their hydrophobic nature. The main objective of this study was to harness surface functionalization technologies to fabricate scaffolds that would be biocompatible and support the adhesion and proliferation of fibroblast cells. The collagen was immobilized on the surface of functionalized PLA via a novel natural cross-linking molecule genipin which resulted in improved cell proliferation of human dermal fibroblasts as compared to the PLA surface coated with collagen without genipin. It is believed that genipin helps reduce steric problems between the functional groups and large protein molecules, and enables immobilized peptide to move more freely in a biological environment.

Adipose-derived stem cells (ASC) are multipotent stem cells that show great potential as a cell source for osteogenic tissue replacements and it is critical to understand the underlying mechanisms of lineage specification. Here we explore the role of primary cilia in human ASC (hASC) differentiation. This study focuses on the chemosensitivity of the primary cilium and the action of its associated proteins: polycystin-1 (PC1), polycystin-2 (PC2) and intraflagellar transport protein-88 (IFT88), in hASC osteogenesis. To elucidate cilia-mediated mechanisms of hASC differentiation, siRNA knockdown of PC1, PC2 and IFT88 was performed to disrupt cilia-associated protein function. Immunostaining of the primary cilium structure indicated phenotypic-dependent changes in cilia morphology. hASC cultured in osteogenic differentiation media yielded cilia of a more elongated conformation than those cultured in expansion media, indicating cilia-sensitivity to the chemical environment and a relationship between the cilium structure and phenotypic determination. Abrogation of PC1, PC2 and IFT88 effected changes in both hASC proliferation and differentiation activity, as measured through proliferative activity, expression of osteogenic gene markers, calcium accretion and endogenous alkaline phosphatase activity. Results indicated that IFT88 may be an early mediator of the hASC differentiation process with its knockdown increasing hASC proliferation and decreasing Runx2, alkaline phosphatase and BMP-2 mRNA expression. PC1 and PC2 knockdown affected later osteogenic gene and end-product expression. PC1 knockdown resulted in downregulation of alkaline phosphatase and osteocalcin gene expression, diminished calcium accretion and reduced alkaline phosphatase enzymatic activity. Taken together our results indicate that the structure of the primary cilium is intimately associated with the process of hASC osteogenic differentiation and that its associated proteins are critical players in this process. Elucidating the dynamic role of the primary cilium and its associated proteins will help advance the application of hASC in generating autologous tissue engineered therapies in critical defect bone injuries.

This study investigates the effects of cyclic hydrostatic pressure (CHP) on chondrogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3-D) agarose constructs maintained in a complete growth medium without soluble chondrogenic inducing factors. hASCs were seeded in 2% agarose hydrogels and exposed to 7.5 MPa CHP for 4 h per day at a frequency of 1 Hz for up to 21 days. On days 0, 7, 14, and 21, the expression levels of collagen II, Sox9, aggrecan, and cartilage oligomeric matrix protein (COMP) were examined by real-time reverse transcriptase-polymerase chain reaction analysis. Gene expression analysis found collagen II mRNA expression in only the CHP-loaded construct at day 14 and at no other time during the study. CHP-loaded hASCs exhibited upregulated mRNA expression of Sox9, aggrecan, and COMP at day 7 relative to unloaded controls, suggesting that CHP initiated chondrogenic differentiation of hASCs in a manner similar to human bone marrow-derived mesenchymal stem cells (hMSC). By day 14, however, loaded hASC constructs exhibited significantly lower mRNA expression of the chondrogenic markers than unloaded controls. Additionally, by day 21, the samples exhibited little measurable mRNA expression at all, suggesting a decreased viability. Histological analysis validated the lack of mRNA expression at day 21 for both the loaded and unloaded control samples with a visible decrease in the cell number and change in morphology. A comparative study with hASCs and hMSCs further examined long-term cell viability in 3-D agarose constructs of both cell types. Decreased cell metabolic activity was observed throughout the 21-day experimental period in both the CHP-loaded and control constructs of both hMSCs and hASCs, suggesting a decrease in cell metabolic activity, alluding to a decrease in cell viability. This suggests that a 2% agarose hydrogel may not optimally support hASC or hMSC viability in a complete growth medium in the absence of soluble chondrogenic inducing factors over long culture durations. This is the first study to examine the ability of mechanical stimuli alone, in the absence of chondrogenic factors transforming growth factor beta (TGF-β)3, TGF-β1 and/or bone morphogenetic protein 6 (BMP6) to induce hASC chondrogenic differentiation. The findings of this study suggest that CHP initiates hASC chondrogenic differentiation, even in the absence of soluble chondrogenic inductive factors, confirming the importance of considering both mechanical stimuli and appropriate 3-D culture for cartilage tissue engineering using hASCs.

Incorporation of mammalian cells into nanofibers (cell electrospinning) and multilayered cell-nanofiber structures (cell layering) via electrospinning are promising techniques for tissue engineering applications. We investigate the viability of 3T3-L1 mouse fibroblasts after incorporation into poly(vinyl alcohol) nanofibers and multilayering with poly(caprolactone) nanofibers and analyze the possible factors that affect cell viability. We observe that cells do not survive cell electrospinning but survive cell layering. Assessing the factors involved in cell electrospinning, we find that dehydration and fiber stretching are the main causes of cell death. In cell layering, the choice of solvent is critical, as residual solvent in the electrospun fibers could be detrimental to the cells.

Human adipose-derived stem cells (hASCs) are a relatively abundant and accessible stem cell source with multilineage differentiation capability and have great potential for bone tissue engineering applications. The success of bone tissue engineering is intimately linked with the production of a mineralized matrix that mimics the natural mineral present within native bone. In this study, we examined the effects of ionic calcium levels of 1.8 (normal concentration in cell culture medium), 8, and 16 mM on hASCs seeded in both two-dimensional monolayer and three-dimensional electrospun scaffolds and cultured in either complete growth medium (CGM) or osteogenic differentiation medium (ODM). The impact of calcium supplementation on hASC viability, proliferation, and mineral deposition was determined. hASCs remained viable for all experimental treatments. hASC proliferation increased with the addition of 8 mM Ca2+ CGM, but decreased for the 16 mM Ca2+ CGM treatment. Materials deposited by hASCs were analyzed using four techniques: (1) histological staining with Alizarin Red S, (2) calcium quantification, (3) Fourier transform infrared spectroscopy, and (4) wide-angle X-ray diffraction. Mineral deposition was significantly enhanced under both growth and osteogenic medium conditions by increasing extracellular Ca2+. The greatest mineral deposition occurred in the ODM 8 mM Ca2+ treatment group. Fourier transform infrared spectroscopy analysis indicated that elevated calcium concentrations of 8 mM Ca2+ significantly increased both PO4 amount and PO4 to protein ratio for ODM. X-ray diffraction indicated that mineral produced with elevated Ca2+ in both CGM and ODM had a crystalline structure characteristic of hydroxyapatite. Ionic calcium should be considered a potent regulator in hASC mineralization and could serve as a potential treatment for inducing prompt ossification of hASC-seeded scaffolds for bone tissue engineering prior to implantation.

To study the potential effects of low-intensity pulsed ultrasound (LIPUS) on cell response in vitro, the ability to alter LIPUS parameters is required. However, commercial LIPUS systems have very little control over parameter selection. In this study, a custom LIPUS system was designed and validated by exploring the effects of using different pulse repetition frequency (PRF) parameters on human adipose derived adult stem cells (hASCs) and bone marrow derived mesenchymal stem cells (hMSCs), two common stem cell sources for creating bone constructs in vitro. Changing the PRF was found to affect cellular response to LIPUS stimulation for both cell types. Proliferation of LIPUS-stimulated cells was found to decrease for hASCs by d 7 for all three groups compared with unstimulated control cells (P = 0.008, 0.011, 0.014 for 1 Hz, 100 Hz and 1 kHz PRF, respectively) and for hMSCs by d 14 (donor 1: P = 0.0005, 0.0002, 0.0003; donor 2: P = 0.0003, 0.0002, 0.0001; for PRFs of 1 Hz, 100 Hz, and 1 kHz, respectively). Additionally, LIPUS was shown to strongly accelerate osteogenic differentiation of hASCs based on amount of calcium accretion normalized by total DNA (P = 0.003, 0.001, 0.003, and 0.032 between control/100 Hz, control/1 kHz, 1 Hz/1 kHz, and 100 Hz/1 kHz pulse repetition frequencies, respectively). These findings promote the study of using LIPUS to induce osteogenic differentiation and further encourage the exploration of LIPUS parameter optimization. The custom LIPUS system was successfully designed to allow extreme parameter variation, specifically PRF, and encourages further studies.

Introduction Primary cilia, discovered over a century ago and considered to be a largely vestigial organelle, are now implicated as critical mechanosensory organelles for the proper function of a variety of mammalian cell types (1,2). We have identified the presence of primary cilia on human adipose derived stem cells (hASC) and we hypothesize that primary cilia act as mechanosensors, playing a critical role in mediating hASC osteogenic differentiation. Previously, our group verified the mechanosensitivity of hASC through observation of enhanced hASC osteogenic differentiation under cyclic tensile strain (3). This study focuses on identifying the mechanism of mechanically mediated differentiation through elucidating the role of primary cilia in this process. To observe changes in the potential primary cilia-mediated differentiation response, epigenetic siRNA knockdown of cilia-specific proteins and physical disruption of the cilia structure, were used to abrogate the primary cilia and remove function. This study may provide important information on how to better optimize the process of creating functional bone tissue from hASC. Materials and Methods Staining. Cells were fixed in 10% formalin for 30 minutes. Primary cilia were identified on hASC, immunostained with mouse monoclonal anti-acetylated tubulin antibody then probed with a fluorescent secondary goat anti-mouse antibody. Alizarin Red stain was added to fixed cells for 3 minutes to analyze calcium accretion. Primary Cilia Silencing. 60 pmol of siRNA (either nontargeting scramble control, Polycystin-1, Polycystin-2 or IFT-88) and 4 μL of Lipofetamine 2000 transfection reagent per well were added to hASC at 60% confluency in 6-well plate in Opti-Mem I transfection media for 4.5 hours and subsequently replaced with growth media. Real-time PCR. RNA was collected from hASC transfected with either polycystin-1 siRNA or Scramble-A control siRNA at 24, 48 and 72 hours after transfection. PCR was used to analyze expression levels between timedependent controls and polycystin-1 knockdown samples. Calcium Assay. hASC were cultured in osteogenic differentiation media for 15 days, primary cilia proteins were silenced at days 0 and 7, and calcium and DNA were collected at day 15. Calcium was quantified using the Calcium LiquiColor® assay and normalized to amount of DNA per condition. Results Primary cilia were present on many hASC, as identified by a dense clustering of acetylated tubulin projecting from the surface of the cell membranes (Fig. 1). Gene expression analysis of primary cilia-associated polycystin-1 (PC-1) showed an ~50% reduction in expression three days after siRNA transfection, as compared to a non-targeting scramble siRNA control, showing sufficient knockdown (data not shown). Serial siRNA transfections sustained reduced primary cilia mRNA knockdown effects for up to 14 days during the osteogenic differentiation process. After 15 days of osteogenic differentiation, hASC exhibited a reduction in calcium accretion with PC-1 and PC-2 knockdown at days 0 and 7 (Fig. 2). Quantitated diminishment of calcium content was consistent with the trend observed with Alizarin Red staining (Fig. 3).

Low intensity pulsed ultrasound (LIPUS) has been used extensively for fracture healing. Many LIPUS parameters have been studied in depth except for pulse repetition frequency (PRF). However, to examine this parameter, a custom LIPUS system needed to be designed since commercial systems have very little control over parameter modification. Specifically, the PRF of commercial systems is typically set to either 100 Hz or 1 kHz and cannot be changed. A custom system was used to explore the effects of PRF on human adipose derived adult stem cells (hASCs) and bone marrow derived mesenchymal stem cells (hMSCs). The PRF was found to significantly affect cellular response to LIPUS stimulation for both cell types. Additionally, LIPUS was shown to strongly accelerate osteogenic differentiation of hASCs based on amount of calcium accretion normalized by total DNA.

We investigated the effects of two types of cyclic tensile strain, continuous and rest inserted, on osteogenic differentiation of human adipose-derived adult stem cells (hASCs). The influence of these mechanical strains was tested on two hASC lines having different mineral deposition potential, with one cell line depositing approximately nine times as much calcium as the other hASC line after 14 days of culture in osteogenic medium on tissue culture plastic. Results showed that both continuous (10% strain, 1 Hz) and rest inserted cyclic tensile strain (10% strain, 1 Hz, 10 s rest after each cycle) regimens increased the amount and rate of calcium deposition for both high and low calcium depositing hASC lines as compared to unstrained controls. The response was similar for both types of tensile strain for a given cell line, however, cyclic tensile strain had a much stronger osteogenic effect on the high calcium depositing hASC line, suggesting that mechanical loading has a greater effect on cell lines that already have an innate ability to produce bone as compared to cell lines that do not. This is the first study to investigate the osteodifferentiation effects of cyclic tensile strain on hASCs and the first to show that both continuous (10%, 1 Hz) and rest inserted (10%, 1 Hz, 10 s rest) cyclic tensile strain accelerate hASC osteodifferentiation and increase calcium accretion.

Development of tissue-engineered bone constructs has recently focused on the use of electrospun composite scaffolds seeded with stem cells from various source tissues. In this study, we fabricated electrospun composite scaffolds consisting of beta-tricalcium phosphate (TCP) crystals and poly(L-lactic acid) (PLA) at varying loading levels of TCP (0, 5, 10, 20 wt%) and assessed the composite scaffolds' material properties and ability to induce proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) in the presence of osteogenic differentiating medium. The electrospun scaffolds all exhibited a nonwoven structure with an interconnected porous network. With the addition of TCP, the fiber diameter increased with each treatment ranging from 503.39 +/- 20.31 nm for 0 wt% TCP to 1267.36 +/- 59.03 nm for 20 wt% TCP. Tensile properties of the composite scaffolds were assessed and the overall tensile strength of the neat scaffold (0 wt% TCP) was 847 +/- 89.43 kPA; the addition of TCP significantly decreased this value to an average of 350.83 +/- 38.57 kPa. As the electrospun composite scaffolds degraded in vitro, TCP was released into the medium with the largest release occurring within the first 6 days. Human ASCs were able to adhere, proliferate and osteogenically differentiate on all scaffold combinations. DNA content increased in a temporal manner for each scaffold over 18 days in culture although for the day 12 timepoint, the 10 wt% TCP scaffold induced the greatest hASC proliferation. Endogenous alkaline phosphatase activity was enhanced on the composite PLA/TCP scaffolds compared to the PLA control particularly by day 18. It was noted that at the highest TCP loading levels of 10 and 20 wt%, there was a dramatic increase in the amount of cell-mediated mineralization compared to the 5 wt% TCP and the neat PLA scaffold. This work suggests that local environment cues provided by the biochemical nature of the scaffold can accelerate the overall osteogenic differentiation of hASCs and encourage rapid ossification.

This chapter describes general protocols for the isolation of human mesenchymal stem cells (hMSCs) from trabecular bone and adipose tissues using adhesion-based methods. These protocols employ standard experimental techniques and can be readily performed in laboratories with several basic equipment items, including biosafety cabinet, humidified CO2 incubator, centrifuge, inverted microscope, and autoclave. Stem cells from adult tissues are attractive materials for cell therapy, gene therapy, and tissue engineering. These cells generally have restricted lineage potential when compared to embryonic stem cells, and this may be advantageous from the standpoint of controlling cell growth and differentiation in certain therapeutic applications. Since the identification of MSCs in bone marrow, cells with similar multilineage potential have been isolated from other tissues, including trabecular bone and adipose tissue. In a mouse model, adipose- and bone marrow– derived MSCs are equally effective in healing skeletal defects; adipose-derived MSCs are also able to heal bone defects in a rat model. Under stimulation by transforming growth factor-beta, bone marrow-derived MSCs expressed more cartilage extracellular matrix proteins and exhibited a more mature chondrogenic phenotype than adipose-derived MSCs. The protocols are intended to provide practical, generally applicable methods for isolating hMSCs from different tissues. They have been used to prepare and characterize hMSCs from over 50 bone and adipose tissue samples.