Combined 3D bioprinting and tissue-specific ECM system reveals the influence of brain matrix on stem cell differentiation.

We have previously shown that human and murine breast extracellular matrix (ECM) can significantly impact cellular behavior, including stem cell fate determination. It has been established that tissue-specific extracellular matrix from the central nervous system has the capacity to support neuronal survival. However, the characterization of its influence on stem cell differentiation and its adaptation to robust 3D culture models is underdeveloped. To address these issues, we combined our 3D bioprinter with hydrogels containing porcine brain extracellular matrix (BMX) to test the influence of the extracellular matrix on stem cell differentiation. Our 3D bioprinting system generated reproducible 3D neural structures derived from mouse embryonic stem cells (mESCs). We demonstrate that the addition of BMX preferentially influences 3D bioprinted mESCs towards neural lineages compared to standard basement membrane (Geltrex/Matrigel) hydrogels alone. Furthermore, we demonstrate that we can transplant these 3D bioprinted neural cellular structures into a mouse's cleared mammary fat pad, where they continue to grow into larger neural outgrowths. Finally, we demonstrate that direct injection of human induced pluripotent stem cells (hiPSCS) and neural stem cells (NSCs) suspended in pure BMX formed neural structures in vivo. Combined, these findings describe a unique system for studying brain ECM/stem cell interactions and demonstrate that BMX can direct pluripotent stem cells to differentiate down a neural cellular lineage without any additional specific differentiation stimuli.

3D bioprinted mammary organoids and tumoroids in human mammary derived ECM hydrogels.

The extracellular matrix (ECM) of tissues is an important mediator of cell function. Moreover, understanding cellular dynamics within their specific tissue context is also important for developmental biology, cancer research, and regenerative medicine. However, robust in vitro models that incorporate tissue-specific microenvironments are lacking. Here we describe a novel mammary-specific culture protocol that combines a self-gelling hydrogel comprised solely of ECM from decellularized rat or human breast tissue with the use of our previously described 3D bioprinting platform. We initially demonstrate that undigested and decellularized mammary tissue can support mammary epithelial and tumor cell growth. We then describe a methodology for generating mammary ECM extracts that can spontaneously gel to form hydrogels. These ECM hydrogels retain unique structural and signaling profiles that elicit differential responses when normal mammary and breast cancer cells are cultured within them. Using our bioprinter, we establish that we can generate large organoids/tumoroids in the all mammary-derived hydrogel. These findings demonstrate that our system allows for growth of organoids/tumoroids in a tissue-specific matrix with unique properties, thus providing a suitable platform for ECM and epithelial/cancer cell studies. STATEMENT OF SIGNIFICANCE: Factors within extracellular matrices (ECMs) are specific to their tissue of origin. It has been shown that tissue specific factors within the mammary gland's ECM have pronounced effects on cellular differentiation and cancer behavior. Understanding the role of the ECM in controlling cell fate has major implications for developmental biology, tissue engineering, and cancer therapy. However, in vitro models to study cellular interactions with tissue specific ECM are lacking. Here we describe the generation of 3D hydrogels consisting solely of human or mouse mammary ECM. We demonstrate that these novel 3D culture substrates can sustain large 3D bioprinted organoid and tumoroid formation. This is the first demonstration of an all mammary ECM culture system capable of sustaining large structural growths.

A 3D bioprinter platform for mechanistic analysis of tumoroids and chimeric mammary organoids.

The normal mammary microenvironment can suppress tumorigenesis and redirect cancer cells to adopt a normal mammary epithelial cell fate in vivo. Understanding of this phenomenon offers great promise for novel treatment and detection strategies in cancer, but current model systems make mechanistic insights into the process difficult. We have recently described a low-cost bioprinting platform designed to be accessible for basic cell biology laboratories. Here we report the use of this system for the study of tumorigenesis and microenvironmental redirection of breast cancer cells. We show our bioprinter significantly increases tumoroid formation in 3D collagen gels and allows for precise generation of tumoroid arrays. We also demonstrate that we can mimic published in vivo findings by co-printing cancer cells along with normal mammary epithelial cells to generate chimeric organoids. These chimeric organoids contain cancer cells that take part in normal luminal formation. Furthermore, we show for the first time that cancer cells within chimeric structures have a significant increase in 5-hydroxymethylcytosine levels as compared to bioprinted tumoroids. These results demonstrate the capacity of our 3D bioprinting platform to study tumorigenesis and microenvironmental control of breast cancer and highlight a novel mechanistic insight into the process of microenvironmental control of cancer.

Correction to: Consistent and reproducible cultures of large-scale 3D mammary epithelial structures using an accessible bioprinting platform.

Following publication of the original article [1], the authors reported a typesetting error in the spelling of the second author's name.

Consistent and reproducible cultures of large-scale 3D mammary epithelial structures using an accessible bioprinting platform.

BACKGROUND: Standard three-dimensional (3D) in vitro culture techniques, such as those used for mammary epithelial cells, rely on random distribution of cells within hydrogels. Although these systems offer advantages over traditional 2D models, limitations persist owing to the lack of control over cellular placement within the hydrogel. This results in experimental inconsistencies and random organoid morphology. Robust, high-throughput experimentation requires greater standardization of 3D epithelial culture techniques. METHODS: Here, we detail the use of a 3D bioprinting platform as an investigative tool to control the 3D formation of organoids through the "self-assembly" of human mammary epithelial cells. Experimental bioprinting procedures were optimized to enable the formation of controlled arrays of individual mammary organoids. We define the distance and cell number parameters necessary to print individual organoids that do not interact between print locations as well as those required to generate large contiguous organoids connected through multiple print locations. RESULTS: We demonstrate that as few as 10 cells can be used to form 3D mammary structures in a single print and that prints up to 500 µm apart can fuse to form single large structures. Using these fusion parameters, we demonstrate that both linear and non-linear (contiguous circles) can be generated with sizes of 3 mm in length/diameter. We confirm that cells from individual prints interact to form structures with a contiguous lumen. Finally, we demonstrate that organoids can be printed into human collagen hydrogels, allowing for all-human 3D culture systems. CONCLUSIONS: Our platform is adaptable to different culturing protocols and is superior to traditional random 3D culture techniques in efficiency, reproducibility, and scalability. Importantly, owing to the low-cost accessibility and computer numerical control-driven platform of our 3D bioprinter, we have the ability to disseminate our experiments with absolute precision to interested laboratories.

ALDH1A1 positive cells are a unique component of the tonsillar crypt niche and are lost along with NGFR positive stem cells during tumourigenesis.

Interest into the cellular biology of human tonsillar crypts has grown in recent years because it is now known to be the site of origin of most human papilloma virus (HPV) induced oropharyngeal squamous cell carcinomas (OPSCC). Despite the interest, still relatively little is known regarding the cellular hierarchy and dynamics of this anatomical subsite. Here we evaluate normal tonsillar crypts for expression of putative stem cell markers. We found that ALDH1A1 was uniquely expressed in a subset of suprabasal tonsillar crypt epithelium. This cell population was unique from NGFR expressing cells, which were previously identified to have stem/progenitor activity in vitro. In vivo mitochondrial lineage tracing was consistent with a basal to luminal progression of cellular development. This provides support for NGFR cells as the resident stem/progenitor cells in tonsillar crypts, and suggests that the ALDH1A1 cells are not stem/progenitor cells, but merely a unique component of the crypt cellular microenvironment. Analysis of tumours found that both NGFR and ALDH1A1 are lost in HPV+ and HPV- tumours, while LGR5 expression is induced in the same tumours. These results identify a unique component of the tonsillar crypt epithelium-ALDH1A1 cells-and support a cellular model where NGFR+ cells are the long-lived progenitor cells within tonsillar crypts. They also provide evidence that NGFR and ALDH1A1+ cells are lost during tumourigenesis.

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.

Huntington's 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.

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.

Preferential Lineage-Specific Differentiation of Osteoblast-Derived Induced Pluripotent Stem Cells into Osteoprogenitors.

While induced pluripotent stem cells (iPSCs) hold great clinical promise, one hurdle that remains is the existence of a parental germ-layer memory in reprogrammed cells leading to preferential differentiation fates. While it is problematic for generating cells vastly different from the reprogrammed cells' origins, it could be advantageous for the reliable generation of germ-layer specific cell types for future therapeutic use. Here we use human osteoblast-derived iPSCs (hOB-iPSCs) to generate induced osteoprogenitors (iOPs). Osteoblasts were successfully reprogrammed and demonstrated by endogenous upregulation of Oct4, Sox2, Nanog, TRA-1-81, TRA-16-1, SSEA3, and confirmatory hPSC Scorecard Algorithmic Assessment. The hOB-iPSCs formed embryoid bodies with cells of ectoderm and mesoderm but have low capacity to form endodermal cells. Differentiation into osteoprogenitors occurred within only 2-6 days, with a population doubling rate of less than 24 hrs; however, hOB-iPSC derived osteoprogenitors were only able to form osteogenic and chondrogenic cells but not adipogenic cells. Consistent with this, hOB-iOPs were found to have higher methylation of PPARγ but similar levels of methylation on the RUNX2 promoter. These data demonstrate that iPSCs can be generated from human osteoblasts, but variant methylation patterns affect their differentiation capacities. Therefore, epigenetic memory can be exploited for efficient generation of clinically relevant quantities of osteoprogenitor cells.

Accessible bioprinting: adaptation of a low-cost 3D-printer for precise cell placement and stem cell differentiation.

The precision and repeatability offered by computer-aided design and computer-numerically controlled techniques in biofabrication processes is quickly becoming an industry standard. However, many hurdles still exist before these techniques can be used in research laboratories for cellular and molecular biology applications. Extrusion-based bioprinting systems have been characterized by high development costs, injector clogging, difficulty achieving small cell number deposits, decreased cell viability, and altered cell function post-printing. To circumvent the high-price barrier to entry of conventional bioprinters, we designed and 3D printed components for the adaptation of an inexpensive 'off-the-shelf' commercially available 3D printer. We also demonstrate via goal based computer simulations that the needle geometries of conventional commercially standardized, 'luer-lock' syringe-needle systems cause many of the issues plaguing conventional bioprinters. To address these performance limitations we optimized flow within several microneedle geometries, which revealed a short tapered injector design with minimal cylindrical needle length was ideal to minimize cell strain and accretion. We then experimentally quantified these geometries using pulled glass microcapillary pipettes and our modified, low-cost 3D printer. This systems performance validated our models exhibiting: reduced clogging, single cell print resolution, and maintenance of cell viability without the use of a sacrificial vehicle. Using this system we show the successful printing of human induced pluripotent stem cells (hiPSCs) into Geltrex and note their retention of a pluripotent state 7 d post printing. We also show embryoid body differentiation of hiPSC by injection into differentiation conducive environments, wherein we observed continuous growth, emergence of various evaginations, and post-printing gene expression indicative of the presence of all three germ layers. These data demonstrate an accessible open-source 3D bioprinter capable of serving the needs of any laboratory interested in 3D cellular interactions and tissue engineering.

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.

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.

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.

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.

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.

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.