Secreted Protein Acidic and Rich in Cysteine (SPARC) Mediates Metastatic Dormancy of Prostate Cancer in Bone.

Prostate cancer is known to frequently recur in bone; however, how dormant cells switch its phenotype leading to recurrent tumor remains poorly understood. We have isolated two syngeneic cell lines (indolent and aggressive) through in vivo selection by implanting PC3mm stem-like cells into tibial bones. We found that indolent cells retained the dormant phenotype, whereas aggressive cells grew rapidly in bone in vivo, and the growth rates of both cells in culture were similar, suggesting a role of the tumor microenvironment in the regulation of dormancy and recurrence. Indolent cells were found to secrete a high level of secreted protein acidic and rich in cysteine (SPARC), which significantly stimulated the expression of BMP7 in bone marrow stromal cells. The secreted BMP7 then kept cancer cells in a dormant state by inducing senescence, reducing "stemness," and activating dormancy-associated p38 MAPK signaling and p21 expression in cancer cells. Importantly, we found that SPARC was epigenetically silenced in aggressive cells by promoter methylation, but 5-azacytidine treatment reactivated the expression. Furthermore, high SPARC promoter methylation negatively correlated with disease-free survival of prostate cancer patients. We also found that the COX2 inhibitor NS398 down-regulated DNMTs and increased expression of SPARC, which led to tumor growth suppression in bone in vivo These findings suggest that SPARC plays a key role in maintaining the dormancy of prostate cancer cells in the bone microenvironment.

Roles of the cyclooxygenase 2 matrix metalloproteinase 1 pathway in brain metastasis of breast cancer.

Brain is one of the major sites of metastasis in breast cancer; however, the pathological mechanism of brain metastasis is poorly understood. One of the critical rate-limiting steps of brain metastasis is the breaching of blood-brain barrier, which acts as a selective interface between the circulation and the central nervous system, and this process is considered to involve tumor-secreted proteinases. We analyzed clinical significance of 21 matrix metalloproteinases on brain metastasis-free survival of breast cancer followed by verification in brain metastatic cell lines and found that only matrix metalloproteinase 1 (MMP1) is significantly correlated with brain metastasis. We have shown that MMP1 is highly expressed in brain metastatic cells and is capable of degrading Claudin and Occludin but not Zo-1, which are key components of blood-brain barrier. Knockdown of MMP1 in brain metastatic cells significantly suppressed their ability of brain metastasis in vivo, whereas ectopic expression of MMP1 significantly increased the brain metastatic ability of the cells that are not brain metastatic. We also found that COX2 was highly up-regulated in brain metastatic cells and that COX2-induced prostaglandins were directly able to promote the expression of MMP1 followed by augmenting brain metastasis. Furthermore, we found that COX2 and prostaglandin were able to activate astrocytes to release chemokine (C-C motif) ligand 7 (CCL7), which in turn promoted self-renewal of tumor-initiating cells in the brain and that knockdown of COX2 significantly reduced the brain metastatic ability of tumor cells. Our results suggest the COX2-MMP1/CCL7 axis as a novel therapeutic target for brain metastasis.

Extracellular vesicles as carriers of microRNA, proteins and lipids in tumor microenvironment.

In recent years, the knowledge about the control of tumor microenvironment has increased and emerged as an important player in tumorigenesis. The role of normal stromal cells in the tumor initiation and progression has brought our vision in to the forefront of cell-to-cell communication. In this review, we focus on the mechanism of communication between stromal and tumor cells, which is based on the exchange of extracellular vesicles (EVs). We describe several, evergrowing, pieces of evidence that EVs transfer messages through their miRNA, lipid, protein and nucleic acid contents. A better understanding of this sophisticated method of communication between normal cancer cells may lead to developing novel approaches for personalized diagnostics and therapeutics.

Quantification of Small Extracellular Vesicles by Size Exclusion Chromatography with Fluorescence Detection.

Chemical analysis of small extracellular vesicles (sEVs) circulating in body fluids holds potentials in noninvasive diagnosis of diseases and evaluation of therapeutic treatments. However, quantification of sEVs remains a challenge due to lacking of cost-effective analytical protocols. Herein we report a facile method based on size exclusion chromatography with fluorescence detection (SEC-FD) for sEVs quantification. After removal of cells and cell debris, a 0.50 mL sample (e.g., cell culture medium) is incubated with CM-Dil dye to fluorescently label sEVs. The incubation solution is then separated on a SEC column packed with Sepharose CL-4B. The eluent is monitored fluorescently at Ex553 nm/Em570 nm by using a fluorometer equipped with a 50-µL flow through cuvette. Separation efficiency of the proposed SEC-FD method was evaluated by analyzing 100 nm liposomes and albumin-FITC conjugate. Liposomes were eluted out in less than 6 min, about 10 min before albumin-FITC. A separation repeatability (RSD in retention time) of 1.4% (n = 5) was obtained for liposomes. In analysis of cell culture media, linear calibration curves based on SEC-FD peak height versus sEVs concentration were obtained with r2 value of 0.996. Intraday quantification repeatability (RSD in peak height) was 3.2% (n = 5). The detection limit was estimated to be 2.9 × 107 exosome particles/mL. The proposed assay was applied to the first study of sEVs secretion from TK6 cells cultured in serum-free medium for a culturing period from 1 to 48 h.

The current landscape of the mesenchymal stromal cell secretome: A new paradigm for cell-free regeneration.

The unique properties of mesenchymal stromal/stem cells (MSCs) to self-renew and their multipotentiality have rendered them attractive to researchers and clinicians. In addition to the differentiation potential, the broad repertoire of secreted trophic factors (cytokines) exhibiting diverse functions such as immunomodulation, anti-inflammatory activity, angiogenesis and anti-apoptotic, commonly referred to as the MSC secretome, has gained immense attention in the past few years. There is enough evidence to show that the one important pathway by which MSCs participate in tissue repair and regeneration is through its secretome. Concurrently, a large body of MSC research has focused on characterization of the MSC secretome; this includes both soluble factors and factors released in extracellular vesicles, for example, exosomes and microvesicles. This review provides an overview of our current understanding of the MSC secretome with respect to their potential clinical applications.

Research using Mesenchymal Stem/Stromal Cells: quality metric towards developing a reference material.

Mesenchymal stem/stromal cells (MSCs) have been extensively investigated for their regenerative, immune-modulatory, and wound healing properties. While the laboratory studies have suggested that MSC's have a unique potential for modulating the etiopathology of multiple diseases, the results from clinical trials have not been encouraging or reproducible. One of the explanations for such variability is explained by the "art" of isolating and propagating MSCs. Therefore, establishing more than minimal criteria to define MSC would help understand best protocols to isolate, propagate and deliver MSCs. Developing a calibration standard, a database and a set of functional tests would be a better quality metric for MSCs. In this review, we discuss the importance of selecting a standard, issues associated with coming up with such a standard and how these issues can be mitigated.

Exosome-mediated transfer of miR-10b promotes cell invasion in breast cancer.

INTRODUCTION: Exosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme encoded by the smpd3 gene that triggers exosome secretion. However, whether exosome-mediated microRNA transfer plays any role in cell invasion remains poorly understood. Thus, the aim of this study was to identify the exosomal microRNAs involved in breast cancer invasion. METHODS: The expression level of endogenous and exosomal miRNAs were examined by real time PCR and the expression level of target proteins were detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study its uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-10b was estimated by invasion assay. RESULTS: In this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes. In particular, nSMase2 or ceramide promotes the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppresses this secretion. Moreover, upon uptake, miR-10b can suppress the protein level of its target genes such as HOXD10 and KLF4, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could induce the invasion ability of non-malignant HMLE cells. CONCLUSION: Together, our results suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through exosomes. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.

The combined effect of encapsulating curcumin and C6 ceramide in liposomal nanoparticles against osteosarcoma.

This study examines the antitumor potential of curcumin and C6 ceramide (C6) against osteosarcoma (OS) cell lines when both are encapsulated in the bilayer of liposomal nanoparticles. Three liposomal formulations were prepared: curcumin liposomes, C6 liposomes and C6-curcumin liposomes. Curcumin in combination with C6 showed 1.5 times enhanced cytotoxic effect in the case of MG-63 and KHOS OS cell lines, in comparison with curcumin liposomes alone. Importantly, C6-curcumin liposomes were found to be less toxic on untransformed primary human cells (human mesenchymal stem cells) in comparison to OS cell lines. In addition, cell cycle assays on a KHOS cell line after treatment revealed that curcumin only liposomes induced G2/M arrest by upregulation of cyclin B1, while C6 only liposomes induced G1 arrest by downregulation of cyclin D1. C6-curcumin liposomes induced G2/M arrest and showed a combined effect in the expression levels of cyclin D1 and cyclin B1. The efficiency of the preparations was tested in vivo using a human osteosarcoma xenograft assay. Using pegylated liposomes to increase the plasma half-life and tagging with folate (FA) for targeted delivery in vivo, a significant reduction in tumor size was observed with C6-curcumin-FA liposomes. The encapsulation of two water insoluble drugs, curcumin and C6, in the lipid bilayer of liposomes enhances the cytotoxic effect and validates the potential of combined drug therapy.

"A novel in vivo model for the study of human breast cancer metastasis using primary breast tumor-initiating cells from patient biopsies".

BACKGROUND: The study of breast cancer metastasis depends on the use of established breast cancer cell lines that do not accurately represent the heterogeneity and complexity of human breast tumors. A tumor model was developed using primary breast tumor-initiating cells isolated from patient core biopsies that would more accurately reflect human breast cancer metastasis. METHODS: Tumorspheres were isolated under serum-free culture conditions from core biopsies collected from five patients with clinical diagnosis of invasive ductal carcinoma (IDC). Isolated tumorspheres were transplanted into the mammary fat pad of NUDE mice to establish tumorigenicity in vivo. Tumors and metastatic lesions were analyzed by hematoxylin and eosin (H+E) staining and immunohistochemistry (IHC). RESULTS: Tumorspheres were successfully isolated from all patient core biopsies, independent of the estrogen receptor α (ERα)/progesterone receptor (PR)/Her2/neu status or tumor grade. Each tumorsphere was estimated to contain 50-100 cells. Transplantation of 50 tumorspheres (1-5 × 103 cells) in combination with Matrigel into the mammary fat pad of NUDE mice resulted in small, palpable tumors that were sustained up to 12 months post-injection. Tumors were serially transplanted three times by re-isolation of tumorspheres from the tumors and injection into the mammary fat pad of NUDE mice. At 3 months post-injection, micrometastases to the lung, liver, kidneys, brain and femur were detected by measuring content of human chromosome 17. Visible macrometastases were detected in the lung, liver and kidneys by 6 months post-injection. Primary tumors variably expressed cytokeratins, Her2/neu, cytoplasmic E-cadherin, nuclear ß catenin and fibronectin but were negative for ERα and vimentin. In lung and liver metastases, variable redistribution of E-cadherin and ß catenin to the membrane of tumor cells was observed. ERα was re-expressed in lung metastatic cells in two of five samples. CONCLUSIONS: Tumorspheres isolated under defined culture conditions from patient core biopsies were tumorigenic when transplanted into the mammary fat pad of NUDE mice, and metastasized to multiple mouse organs. Micrometastases in mouse organs demonstrated a dormancy period prior to outgrowth of macrometastases. The development of macrometastases with organ-specific phenotypic distinctions provides a superior model for the investigation of organ-specific effects on metastatic cancer cell survival and growth.

Somatic expression of LINE-1 elements in human tissues.

LINE-1 expression damages host DNA via insertions and endonuclease-dependent DNA double-strand breaks (DSBs) that are highly toxic and mutagenic. The predominant tissue of LINE-1 expression has been considered to be the germ line. We show that both full-length and processed L1 transcripts are widespread in human somatic tissues and transformed cells, with significant variation in both L1 expression and L1 mRNA processing. This is the first demonstration that RNA processing is a major regulator of L1 activity. Many tissues also produce translatable spliced transcript (SpORF2). An Alu retrotransposition assay, COMET assays and 53BP1 foci staining show that the SpORF2 product can support functional ORF2 protein expression and can induce DNA damage in normal cells. Tests of the senescence-associated beta-galactosidase expression suggest that expression of exogenous full-length L1, or the SpORF2 mRNA alone in human fibroblasts and adult stem cells triggers a senescence-like phenotype, which is one of the reported responses to DNA damage. In contrast to previous assumptions that L1 expression is germ line specific, the increased spectrum of tissues exposed to L1-associated damage suggests a role for L1 as an endogenous mutagen in somatic tissues. These findings have potential consequences for the whole organism in the form of cancer and mammalian aging.

Curcumin-loaded γ-cyclodextrin liposomal nanoparticles as delivery vehicles for osteosarcoma.

The delivery of curcumin, a broad-spectrum anticancer drug, has been explored in the form of liposomal nanoparticles to treat osteosarcoma (OS). Curcumin is water insoluble and an effective delivery route is through encapsulation in cyclodextrins followed by a second encapsulation in liposomes. Liposomal curcumin's potential was evaluated against cancer models of mesenchymal (OS) and epithelial origin (breast cancer). The resulting 2-Hydroxypropyl-γ-cyclodextrin/curcumin - liposome complex shows promising anticancer potential both in vitro and in vivo against KHOS OS cell line and MCF-7 breast cancer cell line. An interesting aspect is that liposomal curcumin initiates the caspase cascade that leads to apoptotic cell death in vitro in comparison with DMSO-curcumin induced autophagic cell death. In addition, the efficiency of the liposomal curcumin formulation was confirmed in vivo using a xenograft OS model. Curcumin-loaded γ-cyclodextrin liposomes indicate significant potential as delivery vehicles for the treatment of cancers of different tissue origin. FROM THE CLINICAL EDITOR: Curcumin-loaded γ-cyclodextrin liposomes were demonstrated in vitro to have significant potential as delivery vehicles for the treatment of cancers of mesenchymal and epithelial origin. Differences between mechanisms of cell death were also evaluated.

Activation of autophagy in mesenchymal stem cells provides tumor stromal support.

Recent studies have implicated multipotential mesenchymal stem cells (MSCs) as an aid to breast cancer cell proliferation and metastasis, partly as a result of the MSCs secretome. As the tumor gets beyond 2 mm in diameter, the stromal cells could undergo starvation due to the lack of sufficient nutrients in solid tumor microenvironment. In this study, we investigated the survival mechanisms used by stressed stromal cells in breast cancers. We used serum-deprived mesenchymal stem cells (SD-MSCs) and MCF-7 breast cancer cells as model system with a hypothesis that stromal cells in the nutrient-deprived core utilize survival mechanisms for supporting surrounding cells. We tested this hypothesis using in vivo tumor xenografts in immunodeficient mice, which indicated that SD-MSCs supported MCF-7 tumor growth by protection from apoptosis. Histochemical assays showed that SD-MSCs-injected tumors exhibited higher cellularity, decreased apoptosis and decreased differentiation. Beclin-1 staining indicated autophagic areas surrounded by actively proliferating cells. Furthermore, in vitro studies demonstrate that SD-MSCs survive using autophagy and secrete paracrine factors that support tumor cells following nutrient/serum deprivation. Western blot and immunocytochemistry analysis of SD-MSCs demonstrated upregulation and perinuclear relocation of autophagy key regulators such as beclin-1, ATG10, ATG12, MAP-LC3 and lysosomes. Electron microscopic analysis detected a time-dependent increase in autophagosome formation and HDAC6 activity assays indicated the upregulation of autophagy. Taken together, these data suggest that under nutrient-deprived conditions that can occur in solid tumors, stromal cells utilize autophagy for survival and also secrete anti-apoptotic factors that can facilitate solid tumor survival and growth.

Human multipotent stromal cells from bone marrow and microRNA: regulation of differentiation and leukemia inhibitory factor expression.

We observed that microRNAs (miRNAs) that regulate differentiation in a variety of simpler systems also regulate differentiation of human multipotent stromal cells (hMSCs) from bone marrow. Differentiation of hMSCs into osteoblasts and adipocytes was inhibited by using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA. Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were up-regulated during osteogenic differentiation and 20 during adipogenic differentiation, 11 of which were commonly up-regulated in both osteogenic and adipogenic differentiation. In silico models predicted that five of the up-regulated miRNAs targeted leukemia inhibitory factor (LIF) expression. The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF.

Adult human mesenchymal stem cells enhance breast tumorigenesis and promote hormone independence.

Adult human mesenchymal stem cells (hMSCs) have been shown to home to sites of breast cancer and integrate into the tumor stroma. We demonstrate here the effect of hMSCs on primary breast tumor growth and the progression of these tumors to hormone independence. Co-injection of bone marrow-derived hMSCs enhances primary tumor growth of the estrogen receptor-positive, hormone-dependent breast carcinoma cell line MCF-7 in the presence or absence of estrogen in SCID/beige mice. We also show hormone-independent growth of MCF-7 cells when co-injected with hMSCs. These effects were found in conjunction with increased immunohistochemical staining of the progesterone receptor in the MCF-7/hMSC tumors as compared to MCF-7 control tumors. This increase in PgR expression indicates a link between MCF-7 cells and MSCs through ER-mediated signaling. Taken together, our data reveal the relationship between tumor microenvironment and tumor growth and the progression to hormone independence. This tumor stroma-cell interaction may provide a novel target for the treatment of estrogen receptor-positive, hormone-independent, and endocrine-resistant breast carcinoma.

Epigenetic reprogramming of IGF1 and leptin genes by serum deprivation in multipotential mesenchymal stromal cells.

Recent studies on the therapeutic effect of multipotential mesenchymal stem cells (MSCs) in various models of injury have shown that paracrine factors secreted by MSCs are responsible for tissue repair with very little engraftment. In this study we tested the hypothesis that MSCs under stress undergo epigenetic modifications that direct secretion of paracrine factors responsible for tissue repair. Microarray assays of MSCs that had been deprived of serum (SD-MSCs), to induce stress, demonstrated an increase in the expression of several angiogenic, prosurvival, and antiapoptotic factors, including insulin-like growth factor 1 (IGF1) and leptin. Real-time polymerase chain reaction assays demonstrated a >200-fold increase in the expression of IGF1 and leptin in SD-MSCs. Chromatin immunoprecipitation of SD-MSCs revealed histone tail modifications consistent with transcriptional activation of IGF1 and leptin promoters in a reversible manner. To identify the functional significance of the epigenetic changes in stressed MSCs, we tested the prosurvival properties of SD-MSCs and the ability of conditioned medium from SD-MSCs to enhance survival of apoptotic cancer cells. First, we showed that SD-MSCs are more resistant to oxidative damage than MSCs using alkaline comet assays. Next, we demonstrated that conditioned medium from SD-MSCs decreased staurosporin-induced cell death in the KHOS osteosarcoma cell line, and that this effect was partially reversed by immunodepletion of IGF1 or leptin from the conditioned medium. In conclusion, we demonstrate that serum deprivation induces epigenetic changes in MSCs to upregulate the expression of the proangiogenic and antiapoptotic factors IGF1 and leptin.

Multipotent stromal cells are activated to reduce apoptosis in part by upregulation and secretion of stanniocalcin-1.

Multipotent stromal cells (MSCs) have been shown to reduce apoptosis in injured cells by secretion of paracrine factors, but these factors were not fully defined. We observed that coculture of MSCs with previously UV-irradiated fibroblasts reduced apoptosis of the irradiated cells, but fresh MSC conditioned medium was unable reproduce the effect. Comparative microarray analysis of MSCs grown in the presence or absence of UV-irradiated fibroblasts demonstrated that the MSCs were activated by the apoptotic cells to increase synthesis and secretion of stanniocalcin-1 (STC-1), a peptide hormone that modulates mineral metabolism and has pleiotrophic effects that have not been fully characterized. We showed that STC-1 was required but not sufficient for reduction of apoptosis of UV-irradiated fibroblasts. In contrast, we demonstrated that MSC-derived STC-1 was both required and sufficient for reduction of apoptosis of lung cancer epithelial cells made apoptotic by incubation at low pH in hypoxia. Our data demonstrate that STC-1 mediates the antiapoptotic effects of MSCs in two distinct models of apoptosis in vitro.

Angiogenic effects of human multipotent stromal cell conditioned medium activate the PI3K-Akt pathway in hypoxic endothelial cells to inhibit apoptosis, increase survival, and stimulate angiogenesis.

Recent reports indicated that vascular remodeling and angiogenesis are promoted by conditioned medium from the cells referred to as multipotent stromal cells (MSCs). However, the molecular events triggered by MSC-conditioned medium (CdM) were not defined. We examined the effects of CdM from human MSCs on cultures of primary human aortic endothelial cells (HAECs). The CdM inhibited hypoxia-induced apoptosis and cell death of HAECs. It also promoted tube formation by HAECs in an assay in vitro. Conditioned medium from multipotent stromal cells incubated under hypoxic conditions in serum-free endothelial basal medium for 2 days (CdM(Hyp)) from hypoxic culture of MSCs was more effective than conditioned medium from MSCs incubated under normoxic conditions in serum-free endothelial basal medium for 2 days from normoxic cultures of MSCs, an observation in part explained by its higher content of antiapoptotic and angiogenic factors, such as interleukin (IL)-6, vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein (MCP)-1. The effects of CdM(Hyp) on hypoxic HAECs were partially duplicated by the addition of IL-6 in a dose-dependent manner; however, anti-IL-6, anti-MCP-1, and anti-VEGF blocking antibodies added independently did not attenuate the effects. Also, addition of CdM(Hyp) activated the PI3K-Akt pathway; the levels of p-Akt and several of its downstream targets were increased by CdM(Hyp), and both the increase in p-Akt and the increase in angiogenesis were blocked by an inhibitor of PI3K-Akt or by expression of a dominant negative gene for PI3K. CdM(Hyp) also increased the levels of p-extracellular signal-regulated kinase (ERK), but there was a minimal effect on p-signal transducer and activator of transcription-3, and an inhibitor of the ERK1/2 pathway had no effect on hypoxia-induced apoptosis of the HAECs. The results are consistent with suggestions that administration of MSCs or factors secreted by MSCs may provide a therapeutic method of decreasing apoptosis and enhancing angiogenesis.

Colony forming unit assays for MSCs.

MSCs are the plastic adherent adult stem/progenitor cells from bone marrow originally referred to as fibroblastoid colony forming units, then in the hematological literature as marrow stromal cells, subsequently as mesenchymal stem cells, and most recently as multipotential mesenchymal stromal cells. MSCs were originally referred to as fibroblastoid colony-forming-cells because one of their characteristic features is adherence to tissue culture plastic and generation of colonies when plated at low densities ( 1,2 ). The efficiency with which they form colonies still remains an important assay for the quality of cell preparations. This chapter describes 2 methods to assay the colony forming ability of MSCs; (a) a traditional assay for colony forming units (CFUs) and (b) single-cell colony forming unit assay (Sc-CFU).

Assays of MSCs with microarrays.

The rapid development of microarray technology during the last decade has greatly expanded the ability to define the genes expressed in cells. This chapter will focus on describing the steps required for conducting successful microarray experiments with multipotential stromal cells (MSCs). A complete microarray experiment, using the Affymetrix system, will be described starting from experimental design and ending with examples of data analysis using the dChip program.

Isolation and culture of bone marrow-derived human multipotent stromal cells (hMSCs).

We have developed protocols whereby a total of 30-90 x 10(6) hMSCs with an average viability greater than 90% can be produced in a single multilevel Cell Factory from a relatively small (1-3 mL) bone marrow aspirate in 14-20 d. It is possible to generate as many as 5 x 10(8) multipotent stromal cells (MSCs) from a single sample, depending on the number of Cell Factories seeded from the initial isolated hMSCs. Briefly, mononuclear cells are collected from a bone marrow aspirate by density gradient centrifugation. The cells are cultured overnight and the adherent cells are allowed to attach to the flask. Nonadherent cells are removed and the culture expanded for 7-10 d with periodic feeding of the cells. The cells are then harvested and seeded at low density (60-100 cells/cm2) into Nunc Cell Factories. The cells are allowed to expand for an additional 7-10 d, and are then harvested.