Native extracellular matrix, synthesized ex vivo by bone marrow or adipose stromal cells, faithfully directs mesenchymal stem cell differentiation.

Mesenchymal stem cells (MSCs) are highly responsive to cues in the microenvironment (niche) that must be recapitulated ex vivo to study their authentic behavior. In this study, we hypothesized that native bone marrow (BM)- and adipose (AD)-derived extracellular matrices (ECM) were unique in their ability to control MSC behavior. To test this, we compared proliferation and differentiation of bone marrow (BM)-derived MSCs when maintained on native decellularized ECM produced by BM versus AD stromal cells (i.e. BM- versus AD-ECM). We found that both ECMs contained similar types of collagens but differed in the relative abundance of each. Type VI collagen was the most abundant (≈60% of the total collagen present), while type I was the next most abundant at ≈30%. These two types of collagen were found in nearly equal proportions in both ECMs. In contrast, type XII collagen was almost exclusively found in AD-ECM, while types IV and V were only found in BM-ECM. Physically and mechanically, BM-ECM was rougher and stiffer, but less adhesive, than AD-ECM. During 14 days in culture, both ECMs supported BM-MSC proliferation better than tissue culture plastic (TCP), although MSC-related surface marker expression remained relatively high on all three culture surfaces. BM-MSCs cultured in osteogenic (OS) differentiation media on BM-ECM displayed a significant increase in calcium deposition in the matrix, indicative of osteogenesis, while BM-MSCs cultured on AD-ECM in the presence of adipogenic (AP) differentiation media showed a significant increase in Oil Red O staining, indicative of adipogenesis. Further, culture on BM-ECM significantly increased BM-MSC-responsiveness to rhBMP-2 (an osteogenic inducer), while culture on AD-ECM enhanced responsiveness to rosiglitazone (an adipogenic inducer). These findings support our hypothesis and indicate that BM- and AD-ECMs retain unique elements, characteristic of their tissue-specific microenvironment (niche), which promote retention of MSC differentiation state (i.e. "stemness") during expansion and direct cell response to lineage-specific inducers. This study provides a new paradigm for precisely controlling MSC fate to a desired cell lineage for tissue-specific cell-based therapies.

Reduced susceptibility to fluconazole in a cat with histoplasmosis.

CASE SUMMARY: An 11-year-old neutered male domestic longhair cat was diagnosed with histoplasmosis from fine-needle aspirates of an abdominal lymph node. Lymph node size initially decreased with fluconazole therapy (11.8 mg/kg PO q12h); however, after 13 months of continuous fluconazole therapy, lymphadenomegaly worsened and samples were collected for culture and antifungal susceptibility. The Histoplasma capsulatum isolate had a very high fluconazole minimum inhibitory concentration (MIC) of 64 µg/ml and an itraconazole MIC of 0.06 µg/ml. The owner declined a change to itraconazole and, ultimately, the cat developed neurologic signs and was euthanized. Owing to the initial response to fluconazole followed by treatment failure and high MIC value, acquired fluconazole resistance was suspected. Clinical breakpoints for fluconazole for the dimorphic fungi are not available to define true antifungal resistance. RELEVANCE AND NOVEL INFORMATION: This is the first published report of reduced susceptibility to fluconazole in a cat being treated for histoplasmosis. Fluconazole failure and increases in MIC between pretreatment and long-term treatment isolates are known to occur in humans with histoplasmosis. Practitioners should be aware of this possibility when treating cats with fluconazole (particularly in cases with long-term [>1 year] fluconazole therapy or in cases with disease recrudescence).

One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior.

For more than 100years, cells and tissues have been studied in vitro using glass and plastic surfaces. Over the last 10-20years, a great body of research has shown that cells are acutely sensitive to their local environment (extracellular matrix, ECM) which contains both chemical and physical cues that influence cell behavior. These observations suggest that modern cell culture systems, using tissue culture polystyrene (TCP) surfaces, may fail to reproduce authentic cell behavior in vitro, resulting in "artificial outcomes." In the current study, we use bone marrow (BM)- and adipose (AD)-derived stromal cells to prepare BM-ECM and AD-ECM, which are decellularized after synthesis by the cells, to mimic the cellular niche for each of these tissues. Each ECM was characterized for its ability to affect BM- and AD-mesenchymal stem cell (MSC) proliferation, as well as proliferation of three cancer cell lines (HeLa, MCF-7, and MDA-MB-231), modulate cell spreading, and direct differentiation relative to standard TCP surfaces. We found that both ECMs promoted the proliferation of MSCs, but that this effect was enhanced when the tissue-origin of the cells matched that of the ECM (i.e. BM-ECM promoted the proliferation of BM-MSCs over AD-MSCs, and vice versa). Moreover, BM- and AD-ECM were shown to preferentially direct MSC differentiation towards either osteogenic or adipogenic lineage, respectively, suggesting that the effects of the ECM were tissue-specific. Further, each ECM influenced cell morphology (i.e. circularity), irrespective of the origin of the MSCs, lending more support to the idea that effects were tissue specific. Interestingly, unlike MSCs, these ECMs did not promote the proliferation of the cancer cells. In an effort to further understand how these three culture substrates influence cell behavior, we evaluated the chemical (protein composition) and physical properties (architecture and mechanical) of the two ECMs. While many structural proteins (e.g. collagen and fibronectin) were found at equivalent levels in both BM- and AD-ECM, the architecture (i.e. fiber orientation; surface roughness) and physical properties (storage modulus, surface energy) of each were unique. These results, demonstrating differences in cell behavior when cultured on the three different substrates (BM- and AD-ECM and TCP) with differences in chemical and physical properties, provide evidence that the two ECMs may recapitulate specific elements of the native stem cell niche for bone marrow and adipose tissues. More broadly, it could be argued that ECMs, elaborated by cells ex vivo, serve as an ideal starting point for developing tissue-specific culture environments. In contrast to TCP, which relies on the "one size fits all" paradigm, native tissue-specific ECM may be a more rational model to approach engineering 3D tissue-specific culture systems to replicate the in vivo niche. We suggest that this approach will provide more meaningful information for basic research studies of cell behavior as well as cell-based therapeutics.

Native extracellular matrix preserves mesenchymal stem cell "stemness" and differentiation potential under serum-free culture conditions.

INTRODUCTION: Bone marrow-derived mesenchymal stem cells (BM-MSCs) for clinical use should not be grown in media containing fetal bovine serum (FBS), because of serum-related concerns over biosafety and batch-to-batch variability. Previously, we described the preparation and use of a cell-free native extracellular matrix (ECM) made by bone marrow cells (BM-ECM) which preserves stem cell properties and enhances proliferation. Here, we compare colony-forming ability and differentiation of MSCs cultured on BM-ECM with a commercially available matrix (CELLstart™) and tissue culture plastic (TCP) under serum-free conditions. METHODS: Primary MSCs from freshly isolated bone marrow-derived mononuclear cells or passaged MSCs (P1) were grown in serum-containing (SCM) or serum-free (SFM) media on BM-ECM, CELLstart™, or TCP substrates. Proliferation, cell composition (phenotype), colony-forming unit replication, and bone morphogenetic protein-2 (BMP-2) responsiveness were compared among cells maintained on the three substrates. RESULTS: Proliferation of primary BM-MSCs was significantly higher in SCM than SFM, irrespectively of culture substrate, suggesting that the expansion of these cells requires SCM. In contrast, passaged cells cultured on BM-ECM or CELLstart™ in SFM proliferated to nearly the same extent as cells in SCM. However, morphologically, those on BM-ECM were smaller and more aligned, slender, and long. Cells grown for 7 days on BM-ECM in SFM were 20-40 % more positive for MSC surface markers than cells cultured on CELLstart™. Cells cultured on TCP contained the smallest number of cells positive for MSC markers. MSC colony-forming ability in SFM, as measured by CFU-fibroblasts, was increased 10-, 9-, and 2-fold when P1 cells were cultured on BM-ECM, CELLstart™, and TCP, respectively. Significantly, CFU-adipocyte and -osteoblast replication of cells grown on BM-ECM was dramatically increased over those on CELLstart™ (2X) and TCP (4-7X). BM-MSCs, cultured in SFM and treated with BMP-2, retained their differentiation capacity better on BM-ECM than on either of the other two substrates. CONCLUSIONS: Our findings indicate that BM-ECM provides a unique microenvironment that supports the colony-forming ability of MSCs in SFM and preserves their stem cell properties. The establishment of a robust culture system, combining native tissue-specific ECM and SFM, provides an avenue for preparing significant numbers of potent MSCs for cell-based therapies in patients.