Patrick C. Sachs

Isolating adipose-derived mesenchymal stem cells from lipoaspirate blood and saline fraction.

Isolation of adipose-derived stem cells (ASCs) typically involves 8+ hours of intense effort, requiring specialized equipment and reagents. Here, we present an improved technique for isolating viable populations of mesenchymal stem cells from lipoaspirate saline fractions within 30 minutes. Importantly, the cells exhibit remarkable similarities to those obtained using the traditional isolation protocols, in terms of their multipotent differentiation potential and immunophenotype. Reducing the acquisition time of ASCs is critical for advancing regenerative medicine therapeutics, and our approach provides rapid and simple techniques for enhanced isolation and expansion of patient-derived mesenchymal stem cells.

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites.

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their “stem cell” characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.

Mesenchymal stem cells in mammary adipose tissue stimulate progression of breast cancer resembling the basal-type

Data are accumulating to support a role for adipose-derived mesenchymal stem cells (MSCs) in breast cancer progression; however, to date most studies have relied on adipose MSCs from non-breast sources. There is a particular need to investigate the role of adipose MSCs in the pathogenesis of basal-like breast cancer, which develops at a disproportionate rate in pre-menopausal African-American women with a gain in adiposity. The aim of this study was to better understand how breast adipose MSCs (bMSCs) contribute to the progression of basal-like breast cancers by relying on isogenic HMT-3255 S3 (pre-invasive) and T4-2 (invasive) human cells that upon transplantation into nude mice resemble this tumor subtype. In vitro results suggested that bMSCs may contribute to breast cancer progression in multiple ways. bMSCs readily penetrate extracellular matrix components in part through their expression of matrix metalloproteinases 1 and 3, promote the invasion of T4-2 cells and efficiently chemoattract endothelial cells via a bFGF-independent, VEGF-A-dependent manner. As mixed xenografts, bMSCs stimulated the growth, invasion and desmoplasia of T4-2 tumors, yet these resident stem cells showed no observable effect on the progression of pre-invasive S3 cells. While bMSCs form vessel-like structures within Matrigel both in vitro and in vivo and chemoattract endothelial cells, there appeared to be no difference between T4-2/bMSC mixed xenografts and T4-2 xenografts with regard to intra- or peri-tumoral vascularity. Collectively, our data suggest that bMSCs may contribute to the progression of basal-like breast cancers by stimulating growth and invasion but not vasculogenesis or angiogenesis.

Electrospinning adipose tissue‐derived extracellular matrix for adipose stem cell culture

Basement membrane-rich extracellular matrices, particularly murine sarcoma-derived Matrigel, play important roles in regenerative medicine research, exhibiting marked cellular responses in vitro and in vivo, although with limited clinical applications. We find that a human-derived matrix from lipoaspirate fat, a tissue rich in basement membrane components, can be fabricated by electrospinning and used to support cell culture. We describe practical applications and purification of extracellular matrix (ECM) from adipose tissue (At-ECM) and its use in electrospinning scaffolds and adipose stem cell (ASC) culture. The matrix composition of this purified and electrospun At-ECM was assessed histochemically for basement membrane, connective tissue, collagen, elastic fibers/elastin, glycoprotein, and proteoglycans. Each histochemical stain was positive in fat tissue, purified At-ECM, and electrospun At-ECM, and to some extent positive in a 10:90 blend with polydioxanone (PDO). We also show that electrospun At-ECM, alone and blended with PDO, supports ASC attachment and growth, suggesting that electrospun At-ECM scaffolds support ASC cultivation. These studies show that At-ECM can be isolated and electrospun as a basement membrane-rich tissue engineering matrix capable of supporting stem cells, providing the groundwork for an array of future regenerative medicine advances.

Genetic inhibition of telomerase results in sensitization and recovery of breast tumor cells

Telomerase, a ribonucleoprotein enzyme minimally composed of an RNA template (human telomerase RNA) and a catalytically active protein subunit (human telomerase reverse transcriptase), synthesizes telomeric repeats onto chromosome ends and is obligatory for continuous tumor cell proliferation. Telomerase is an attractive anticancer therapeutic target because its activity is present in >90% of human cancers, including >95% of breast carcinomas. Traditional chemotherapies lack the ability to effectively control and cure breast cancer, in part because residual cells are often resistant to DNA-damaging modalities. Although numerous telomerase inhibition strategies cause cancer cells to undergo apoptosis or senescence, there is often a lag period between the beginning of the treatment regimen and a biological effect. Thus, our goal for these studies was to show that effectively blocking telomerase genetically together with standard chemotherapeutic agents, doxorubicin/Adriamycin or Taxol, would increase the sensitization and efficacy for triggering senescence and/or apoptosis in cultures of breast cancer cells while reducing toxicity. We find that blocking telomerase in breast tumor cells substantially increases the sensitization at lower doses of Adriamycin or Taxol and that the kinetics of senescence/apoptosis is more rapid at higher concentrations. Combined with telomerase inhibition, Taxol treatment induced both apoptosis (its typical cell fate) and senescence, both at high enough levels to suggest that these two cellular responses are not mutually exclusive. Genetic inhibition of telomerase is eventually reversed due to up-regulation of endogenous telomerase activity without a net change in telomere length, suggesting that telomerase inhibition itself, not necessarily short telomeres, is important for sensitization.[Mol Cancer Ther 2009;8(5):1319–27]

DNA Methylation Leads to DNA Repair Gene Down-Regulation and Trinucleotide Repeat Expansion in Patient-Derived Huntington Disease Cells

Huntington disease (HD) is an autosomal dominantly inherited disease that exhibits genetic anticipation of affected progeny due to expansions of a trinucleotide repeat (TNR) region within the HTT gene. DNA repair machinery is a known effector of TNR instability; however, the specific defects in HD cells that lead to TNR expansion are unknown. We hypothesized that HD cells would be deficient in DNA repair gene expression. To test this hypothesis, we analyzed expression of select DNA repair genes involved in mismatch/loop-out repair (APEX1, BRCA1, RPA1, and RPA3) in patient-derived HD cells and found each was consistently down-regulated relative to wild-type samples taken from unaffected individuals in the same family. Rescue of DNA repair gene expression by 5-azacytidine treatment identified DNA methylation as a mediator of DNA repair gene expression deficiency. Bisulfite sequencing confirmed hypermethylation of the APEX1 promoter region in HD cells relative to control, as well as 5-azacytidine-induced hypomethylation. 5-Azacytidine treatments also resulted in stabilization of TNR expansion within the mutant HTT allele during long-term culture of HD cells. Our findings indicate that DNA methylation leads to DNA repair down-regulation and TNR instability in mitotically active HD cells and offer a proof of principle that epigenetic interventions can curb TNR expansions.

Tissue specific microenvironments: a key tool for tissue engineering and regenerative medicine

The accumulated evidence points to the microenvironment as the primary mediator of cellular fate determination. Comprised of parenchymal cells, stromal cells, structural extracellular matrix proteins, and signaling molecules, the microenvironment is a complex and synergistic edifice that varies tissue to tissue. Furthermore, it has become increasingly clear that the microenvironment plays crucial roles in the establishment and progression of diseases such as cardiovascular disease, neurodegeneration, cancer, and ageing. Here we review the historical perspectives on the microenvironment, and how it has directed current explorations in tissue engineering. By thoroughly understanding the role of the microenvironment, we can begin to correctly manipulate it to prevent and cure diseases through regenerative medicine techniques.

Modeling early stage bone regeneration with biomimetic electrospun fibrinogen nanofibers and adipose-derived mesenchymal stem cells

Abstract The key events of the earliest stages of bone regeneration have been described in vivo although not yet modeled in an in vitro environment, where mechanistic cell-matrix-growth factor interactions can be more effectively studied. Here, we explore an early-stage bone regeneration model where the ability of electrospun fibrinogen (Fg) nanofibers to regulate osteoblastogenesis between distinct mesenchymal stem cells populations is assessed. Electrospun scaffolds of Fg, polydioxanone (PDO), and a Fg:PDO blend were seeded with adipose-derived mesenchymal stem cells (ASCs) and grown for 7-21 days in osteogenic differentiation media or control growth media. Scaffolds were analyzed weekly for histologic and molecular evidence of osteoblastogenesis. In response to osteogenic differentiation media, ASCs seeded on the Fg scaffolds exhibit elevated expression of multiple genes associated with osteoblastogenesis. Histologic stains and scanning electron microscopy demonstrate widespread mineralization within the scaffolds, as well as de novo type I collagen synthesis. Our data demonstrates that electrospun Fg nanofibers support ASC osteogenic differentiation, yet the scaffold itself does not appear to be osteoinductive. Together, ASCs and Fg recapitulate early stages of bone regeneration ex vivo and presents a prospective autologous therapeutic approach for bone repair.

3D bioprinter applied picosecond pulsed electric fields for targeted manipulation of proliferation and lineage specific gene expression in neural stem cells

OBJECTIVE Picosecond pulse electric fields (psPEF) have the potential to elicit functional changes in mammalian cells in a non-contact manner. Such electro-manipulation of pluripotent and multipotent cells could be a tool in both neural interface and tissue engineering. Here, we describe the potential of psPEF in directing neural stem cells (NSCs) gene expression, metabolism, and proliferation. As a comparison mesenchymal stem cells (MSCs) were also tested. APPROACH A psPEF electrode was anchored on a customized commercially available 3D printer, which allowed us to deliver pulses with high spatial precision and systematically control the electrode position in three-axes. When the electrodes are continuously energized and their position is shifted by the 3D printer, large numbers of cells on a surface can be exposed to a uniform psPEF. With two electric field strengths (20 and 40 kV cm-1), cell responses, including cell viability, proliferation, and gene expression assays, were quantified and analyzed. MAIN RESULTS Analysis revealed both NSCs and MSCs showed no significant cell death after treatments. Both cell types exhibited an increased metabolic reduction; however, the response rate for MSCs was sensitive to the change of electric field strength, but for NSCs, it appeared independent of electric field strength. The change in proliferation rate was cell-type specific. MSCs underwent no significant change in proliferation whereas NSCs exhibited an electric field dependent response with the higher electric field producing less proliferation. Further, NSCs showed an upregulation of glial fibrillary acidic protein (GFAP) after 24 h to 40 kV cm-1, which is characteristic of astrocyte specific differentiation. SIGNIFICANCE Changes in cell metabolism, proliferation, and gene expression after picosecond pulsed electric field exposure are cell type specific.

Epigenetic alterations mediate iPSC-induced normalization of DNA repair gene expression and TNR stability in Huntington's disease cells

ABSTRACT Huntingtons disease (HD) is a rare autosomal dominant neurodegenerative disorder caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat (TNR) expansion within the HTT gene. The mechanisms underlying HD-associated cellular dysfunction in pluripotency and neurodevelopment are poorly understood. We had previously identified downregulation of selected DNA repair genes in HD fibroblasts relative to wild-type fibroblasts, as a result of promoter hypermethylation. Here, we tested the hypothesis that hypomethylation during cellular reprogramming to the induced pluripotent stem cell (iPSC) state leads to upregulation of DNA repair genes and stabilization of TNRs in HD cells. We sought to determine how the HD TNR region is affected by global epigenetic changes through cellular reprogramming and early neurodifferentiation. We find that early stage HD-affected neural stem cells (HD-NSCs) contain increased levels of global 5-hydroxymethylation (5-hmC) and normalized DNA repair gene expression. We confirm TNR stability is induced in iPSCs, and maintained in HD-NSCs. We also identify that upregulation of 5-hmC increases ten-eleven translocation 1 and 2 (TET1/2) protein levels, and show their knockdown leads to a corresponding decrease in the expression of select DNA repair genes. We further confirm decreased expression of TET1/2-regulating miR-29 family members in HD-NSCs. Our findings demonstrate that mechanisms associated with pluripotency induction lead to a recovery in the expression of select DNA repair gene and stabilize pathogenic TNRs in HD. Summary: Cellular reprogramming of Huntingtons disease cells to pluripotency causes upregulation of DNA repair genes and stabilizes trinucleotide repeats. The process is mediated by an increase in hydroxymethylation pathways.