Donald G. Phinney

Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair--current views.

Mesenchymal stem cells or multipotent stromal cells (MSCs) isolated from the bone marrow of adult organisms were initially characterized as plastic adherent, fibroblastoid cells with the capacity to generate heterotopic osseous tissue when transplanted in vivo. In recent years, MSCs or MSC‐like cells have been shown to reside within the connective tissue of most organs, and their surface phenotype has been well described. A large number of reports have also indicated that the cells possess the capacity to transdifferentiate into epithelial cells and lineages derived from the neuroectoderm. The broad developmental plasticity of MSCs was originally thought to contribute to their demonstrated efficacy in a wide variety of experimental animal models of disease as well as in human clinical trials. However, new findings suggest that the ability of MSCs to alter the tissue microenvironment via secretion of soluble factors may contribute more significantly than their capacity for transdifferentiation in tissue repair. Herein, we critically evaluate the literature describing the plasticity of MSCs and offer insight into how the molecular and functional heterogeneity of this cell population, which reflects the complexity of marrow stroma as an organ system, may confound interpretation of their transdifferentiation potential. Additionally, we argue that this heterogeneity also provides a basis for the broad therapeutic efficacy of MSCs.

MESENCHYMAL STEM CELL ENGRAFTMENT IN LUNG IS ENHANCED IN RESPONSE TO BLEOMYCIN EXPOSURE AND AMELIORATES ITS FIBROTIC EFFECTS

Previously we described a reliable method based on immunodepletion for isolating mesenchymal stem cells (MSCs) from murine bone marrow and showed that, after intracranial transplantation, the cells migrated throughout forebrain and cerebellum and adopted neural cell fates. Here we systemically administered MSCs purified by immunodepletion from male bleomycin (BLM)-resistant BALB/c mice into female BLM-sensitive C57BL/6 recipients and quantified engraftment levels in lung by real-time PCR. Male DNA accounted for 2.21 × 10-5% of the total lung DNA in control-treated mice but was increased 23-fold (P = 0.05) in animals exposed to BLM before MSC transplantation. Fluorescence in situ hybridization revealed that engrafted male cells were localized to areas of BLM-induced injury and exhibited an epithelium-like morphology. Moreover, purification of type II epithelial cells from the lungs of transplant recipients resulted in a 3-fold enrichment of male, donor-derived cells as compared with whole lung tissue. MSC administration immediately after exposure to BLM also significantly reduced the degree of BLM-induced inflammation and collagen deposition within lung tissue. Collectively, these studies demonstrate that murine MSCs home to lung in response to injury, adopt an epithelium-like phenotype, and reduce inflammation and collagen deposition in lung tissue of mice challenged with BLM.

Propagation and senescence of human marrow stromal cells in culture: a simple colony‐forming assay identifies samples with the greatest potential to propagate and differentiate

Marrow stromal cells (MSCs) were isolated from bone marrow obtained by aspirates of the iliac crest of normal volunteers. The cells were isolated by their adherence to plastic and then passed in culture. Some of the samples expanded through over 15 cell doublings from the time frozen stocks were prepared. Others ceased replicating after about four cell doublings. The replicative potential of the cells in culture was best predicted by a simple colony‐forming assay in which samples from early passages were plated at low densities of about 10 cells per cm2. Samples with high colony‐forming efficiency exhibited the greatest replicative potential. The colonies obtained by plating early passage cells at low density varied in size and morphology. The large colonies readily differentiated into osteoblasts and adipocytes when incubated in the appropriate medium. As samples were expanded in culture and approached senescence, they retained their ability to differentiate into osteoblasts. However, the cells failed to differentiate into adipocytes. The loss of multipotentiality following serial passage in culture may have important implications for the use of expanded MSCs for cell and gene therapy.

Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury

Mesenchymal stem cells (MSCs) have been exploited as cellular vectors to treat a wide array of diseases but the mechanisms responsible for their therapeutic effect remain indeterminate. Previously, we reported that MSCs inhibit bleomycin (BLM)-induced inflammation and fibrosis within the lungs of mice. Interrogation of the MSC transcriptome identified interleukin 1 receptor antagonist (IL1RN) as a potential mediator of this effect. Fractionation studies indicated that MSCs are the principal source of IL1RN in murine bone marrow and that its expression is restricted to a unique subpopulation of cells. Moreover, MSC-conditioned media was shown to block proliferation of an IL-1α-dependent T cell line and inhibit production of TNF-α by activated macrophages in vitro. Studies conducted in mice revealed that MSC administration was more effective than recombinant IL1RN delivered via adenoviral infection or osmotic pumps in inhibiting BLM-induced increases in TNF-α, IL-1α, and IL1RN mRNA in lung, IL1RN protein in bronchoalveolar lavage (BAL) fluid, and trafficking of lymphocytes and neutrophils into the lung. Therefore, MSCs protect lung tissue from BLM-induced injury by blocking TNF-α and IL-1, two fundamental proinflammatory cytokines in lung. Identification of IL1RN-expressing human MSC subpopulations may provide a novel cellular vector for treating chronic inflammatory diseases in humans.

Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: Variations in yield, growth, and differentiation

Bone marrow stroma contains a unique cell population, referred to as marrow stromal cells (MSCs), capable of differentiating along multiple mesenchymal cell lineages. A standard liquid culture system has been developed to isolate MSCs from whole marrow by their adherence to plastic wherein the cells grow as clonal populations derived from a single precursor termed the colony‐forming‐unit fibroblast (CFU‐F). Using this liquid culture system, we demonstrate that the relative abundance of MSCs in the bone marrow of five commonly used inbred strains of mice varies as much as 10‐fold, and that the cells also exhibit markedly disparate levels of alkaline phosphatase expression, an early marker of osteoblast differentiation. For each strain examined, the method of isolating MSCs by plastic adherence yields a heterogeneous cell population. These plastic adherent cells also exhibit widely varying growth kinetics between the different strains. Importantly, of three inbred strains commonly used to prepare transgenic mice that we examined, only cells derived from FVB/N marrow readily expand in culture. Further analysis of cultures derived from FVB/N marrow showed that most plastic adherent cells express CD11b and CD45, epitopes of lymphohematopoietic cells. The later consists of both pre‐B‐cell progenitors, granulocytic and monocytic precursors, and macrophages. However, a subpopulation of the MSCs appear to represent bona fide mesenchymal progenitors, as cells can be induced to differentiate into osteoblasts and adipocytes after exposure to dexamethasone and into myoblasts after exposure to amphotericin B. Our results point to significant strain differences in the properties of MSCs and indicate that standard methods cannot be applied to murine bone marrow to isolate relatively pure populations of MSCs. J. Cell. Biochem. 72:570–585, 1999.

Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis.

Mesenchymal stem cells (MSCs) transplanted at sites of nerve injury are thought to promote functional recovery by producing trophic factors that induce survival and regeneration of host neurons. To evaluate this phenomenon further, we quantified in human MSCs neurotrophin expression levels and their effects on neuronal cell survival and neuritogenesis. Screening a human MSC cDNA library revealed expressed transcripts encoding BDNF and beta-NGF but not NT-3 and NT-4. Immunostaining demonstrated that BDNF and beta-NGF proteins were restricted to specific MSC subpopulations, which was confirmed by ELISA analysis of 56 separate subclones. Using a co-culture assay, we also demonstrated that BDNF expression levels correlated with the ability of MSC populations or subclones to induce survival and neurite outgrowth in the SH-SY5Y neuroblastoma cell line. However, these MSC-induced effects were only partially inhibited by a neutralizing anti-BDNF antibody. MSCs were also shown to promote neurite outgrowth within dorsal root ganglion explants despite secreting 25-fold lower level of beta-NGF required exogenously to produce a similar effect. Interrogation of the human MSC transcriptome identified expressed mRNAs encoding various neurite-inducing factors, axon guidance and neural cell adhesion molecules. Moreover, a subset of these transcripts was shown to correlate with BDNF expression in MSC subclones. Collectively, these studies reveal the existence of MSC subpopulations that co-express neurotrophins and other potent neuro-regulatory molecules, which contribute to MSC-induced effects on neuronal cell survival and nerve regeneration. These subpopulations may represent more potent vectors for treating a variety of neurological disorders.

Characterization of Mesenchymal Stem Cells Isolated From Murine Bone Marrow by Negative Selection

Mesenchymal stem cells (MSCs) are typically enriched from bone marrow via isolation of the plastic adherent, fibroblastoid cell fraction. However, plastic adherent cultures elaborated from murine bone marrow are an admixture of fibroblastoid and hematopoietic cell types. Here we report a reliable method based on immunodepletion to fractionate fibroblastoid cells from hematopoietic cells within plastic adherent murine marrow cultures. The immunodepleted cells expressed the antigens Sca‐1, CD29, CD44, CD81, CD106, and the stem cell marker nucleostemin (NST) but not CD11b, CD31, CD34, CD45, CD48, CD90, CD117, CD135, or the transcription factor Oct‐4. They were also capable of differentiating into adipocytes, chondrocytes, and osteoblasts in vitro as well as osteoblasts/osteocytes in vivo. Therefore, immunodepletion yields a cell population devoid of hematopoietic and endothelial cells that is phenotypically and functionally equivalent to MSCs. The immunodepleted cells exhibited a population doubling time of approximately 5–7 days in culture. Poor growth was due to the dramatic down regulation of many genes involved in cell proliferation and cell cycle progression as a result of immunodepletion. Exposure of immunodepleted cells to fibroblast growth factor 2 (FGF2) but not insulin‐like growth factor (IGF), murine stem cell factor, or leukemia inhibitory factor (LIF) significantly increased their growth rate. Moreover, 82% of the transcripts down regulated by immunodepletion remain unaltered in the presence of FGF2. Exposure to the later also reversibly inhibited the ability of the immunodepleted cells to differentiate into adipocytes, chondrocytes, and osteoblasts in vitro. Therefore, FGF2 appears to function as a mitogen and self‐maintenance factor for murine MSCs enriched from bone marrow by negative selection.

Donor variation in the growth properties and osteogenic potential of human marrow stromal cells.

Human marrow stromal cells (MSCs) were isolated from posterior illiac crest marrow aspirates obtained from 17 healthy donors, ages 19–45 years, with no apparent physical disability. First passage hMSCs exhibited growth rates in vitro that varied up to 12‐fold between donors. No correlation between growth rate and the age or gender of the donor was evident (P ≤ 0.05). When hMSCs were cultured without passage for eight days (subconfluent cultures) or 22 days (confluent cultures) in the absence of any osteogenic agonists, levels of alkaline phosphatase enzyme activity varied 40‐fold and 10‐fold, respectively, between donors. When exposed to osteo‐inductive media, donor populations also showed dramatic differences in levels of bone‐specific gene induction. Collectively, these data demonstrate that hMSC cultures are composed of a heterogeneous mixture of cells at various stages of differentiation and with distinct osteogenic potentials. Differences in both growth rate and ALP activity were evident in hMSC cultures established from multiple aspirates obtained over a six month period from the same donors. Therefore, it appears that cellular heterogeneity produced by the method of harvest is propagated within and among different donor populations during culture expansion in vitro. J. Cell. Biochem. 75:424–436, 1999.

In Vitro High‐Capacity Assay to Quantify the Clonal Heterogeneity in Trilineage Potential of Mesenchymal Stem Cells Reveals a Complex Hierarchy of Lineage Commitment

In regenerative medicine, bone marrow is a promising source of mesenchymal stem cells (MSCs) for a broad range of cellular therapies. This research addresses a basic prerequisite to realize the therapeutic potential of MSCs by developing a novel high‐capacity assay to quantify the clonal heterogeneity in potency that is inherent to MSC preparations. The assay utilizes a 96‐well format to (1) classify MSCs according to colony‐forming efficiency as a measure of proliferation capacity and trilineage potential to exhibit adipo‐, chondro‐, and osteogenesis as a measure of multipotency and (2) preserve a frozen template of MSC clones of known potency for future use. The heterogeneity in trilineage potential of normal bone marrow MSCs is more complex than previously reported: all eight possible categories of trilineage potential were detected. In this study, the average colony‐forming efficiency of MSC preparations was 55–62%, and tripotent MSCs accounted for nearly 50% of the colony‐forming cells. The multiple phenotypes detected in this study infer a more convoluted hierarchy of lineage commitment than described in the literature. Greater cell amplification, colony‐forming efficiency, and colony diameter for tri‐ versus unipotent clones suggest that MSC proliferation may be a function of potency. CD146 may be a marker of multipotency, with ∼2‐fold difference in mean fluorescence intensity between tri‐ and unipotent clones. The significance of these findings is discussed in the context of the efficacy of MSC therapies. The in vitro assay described herein will likely have numerous applications given the importance of heterogeneity to the therapeutic potential of MSCs. STEM CELLS 2010;28:788–798

MicroSAGE analysis of 2,353 expressed genes in a single cell-derived colony of undifferentiated human mesenchymal stem cells reveals mRNAs of multiple cell lineages

Mesenchymal stem cells (MSCs) isolated from the bone marrow of adult organisms are capable of differentiating into adipocytes, chondrocytes, myoblasts, osteoblasts, and hematopoiesis‐supporting stroma. We recently demonstrated that MSCs also adopt glial cell fates when transplanted into the developing central nervous system and hence can produce tissue elements derived from a separate embryonic layer. Despite these remarkable properties, it has been difficult to establish specific criteria to characterize MSCs. Using a modified protocol for micro‐serial analysis of gene expression, we cataloged 2,353 unique genes expressed by a single cell‐derived colony of undifferentiated human MSCs. This analysis revealed that the MSC colony simultaneously expressed transcripts characteristic of various mesenchymal cell lineages including chondrocytes, myoblasts, osteoblasts, and hematopoiesis‐supporting stroma. Therefore, the profile of expressed transcripts reflects the developmental potential of the cells. Additionally, the MSC colony expressed mRNAs characteristic of endothelial, epithelial and neuronal cell lineages, a combination that provides a unique molecular signature for the cells. Other expressed transcripts included various products involved in wound repair as well as several neurotrophic factors. A total of 268 novel transcripts were also identified, one of which is the most abundantly expressed mRNA in MSCs. This study represents the first extensive gene expression analysis of MSCs and as such reveals new insight into the biology, ontogeny, and in vivo function of the cells.