Influence of Angiopoietin Treatment with Hypoxia and Normoxia on Human Intervertebral Disc Progenitor Cell’s Proliferation, Metabolic Activity, and Phenotype

Increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor to low back pain. In addition to a series of pathogenic processes, degenerated IVDs become vascularized in contrast to healthy IVDs. In this context, angiopoietin (Ang) plays a crucial role and is involved in cytokine recruitment, and anabolic and catabolic reactions within the extracellular matrix (ECM). Over the last decade, a progenitor cell population has been described in the nucleus pulposus (NP) of the IVD to be positive for the Tie2 marker (also known as Ang-1 receptor). In this study, we investigated the influence of Ang-1 and Ang-2 on human NP cell (Tie2+, Tie2- or mixed) populations isolated from trauma patients during 7 days in normoxia (21% O2) or hypoxia (≤ 5% O2). At the end of the process, the proliferation and metabolic activity of the NP cells were analyzed. Additionally, the relative gene expression of NP-related markers was evaluated. NP cells showed a higher proliferation depending on the Ang treatment. Moreover, the study revealed higher NP cell metabolism when cultured in hypoxia. Additionally, the relative gene expression followed, with an increase linked to the oxygen level and Ang concentration. Our study comparing different NP cell populations may be the start of new approaches for the treatment of IVD degeneration

Spheroid-like Cultures for Cell Expansion of Angiopoietin Receptor-1 (aka. Tie2) positive Cells from the human Intervertebral Disc

INTRODUCTION:Low back pain is the leading cause of disability worldwide (1). Nevertheless, the mechanism of the intervertebral disc (IVD) degeneration is still not clear. In this context, the nucleus pulposus (NP) and more precisely NP progenitor cells (NPPCs) present in the IVD, positive for angiopoietin-1 receptor (aka. Tie2) display multipotent and stem capacity (2,3). In this study, the first aim was to determine whether spheroid formation in suspension-culture will increase the amount/percentage of NPPCs during the expansion compared to traditional monolayer culture. The second aim of this study was to investigate if the percentage of NPPCs will be enriched even further by the resuspension of the spheroid-like cultured cells (=1st generation) and reformation of those spheroids one more time (= 2nd generation). METHODS:Human NP tissues from trauma patients (N=3) were obtained with written ethical consent and isolated by a two-step digestion protocol (3). The NP cells were resuspended and frozen at -150°C after reaching confluence of passage 0. At passage 1, NP cells were seeded in standard or ultra-low attachment tissue culture flasks with 2.5 ng/ml FGF-2 in low glucose - DMEM (supplemented with 10 % FBS). Flow cytometry was used to analyze and quantify the percentage of NPPCs using Tie2 antibody. We defined the spheroids formed after passage 1 NPCs as 1st generation spheroid. We obtained the 2nd generation spheroids by resuspending the 1st-generation-spheroid and reassembly. The NPCs from 1st and 2nd spheroid were quantified by CFU-assay. RESULTS:As a result, the percentage of NPPCs in monolayer culture condition was reaching 7 ± 2 % (Mean±SEM), however, in the 1st and 2nd generation spheroids culture condition, we were observing 20 ± 10 % and 28 ± 6% of Tie2+ cells, respectively. Concerning the CFU-assay, the NPCs from the 2nd generation spheroid formed 30 CFU-S per 1,000 cells, which were twice more CFU-S compared to the 1st generation spheroid. DISCUSSION & CONCLUSIONS:From these data we conclude than the spheroid-like formation of NPCs would be a more efficient method for expansion and enrichment of NPPCs than monolayer expansion in a context of future cell therapy. Acknowledgements:Financial support was received from iPSpine H2020 project under grant agreement #825925 and China Scholarship Council to X.Z

Mimicking the Intervertebral Disc Microenvironment for Expansion of Nucleus Pulposus Progenitor Cells in a Context of Cell Therapy

INTRODUCTION: Low back pain (LBP) is a global health concern that affects as many as 75–80% of people during their lifetime. Although the causes of LBP are multifactorial, increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor. In this respect, tissue-specific progenitors may play a crucial role in tissue regeneration, as these cells are perfectly adapted to their niche. Recently, the progenitor cell population was described in the nucleus pulposus (NP) of the IVD. These cells, positive for the Tie2 marker, have self-renewal capacity and in vitro multipotency potential. However, extremely low numbers of the NP progenitors limit the feasibility of cell therapy strategies. Here, we study the influence of the culture method and of the microenvironment on the human NP progenitors and their differentiation potential in vitro. METHODS:Cells were obtained from human NP tissue from trauma patients undergoing spinal surgery. Briefly, after mild overnight digestion, the NP tissue cells were cultured in 2D (monolayer) or 3D (alginate beads) conditions with medium supplemented in ascorbic acid. After 2 weeks, cells from 2D or 3D culture were expanded on fibronectin-coating flasks with medium supplemented in FGF-2 to mimic the native microenvironment of NP cells. Subsequently, expanded NP cells were then characterized by cytometry (CD105, CD90, CD73, CD45, CD34, and Tie2) and tri-lineage differentiation, which was analyzed by qPCR and histology. RESULTS: Cytometry analysis, after 2D- or 3D-expansion showed the presence of 0.1 % and 78.2 % of Tie2+ NP progenitors, respectively. Concerning the chondrogenic differentiation assay, the detection of glycosaminoglycans in the culture medium was drastically increased for 3D-expanded cells (11-fold) vs 2D-expanded cells. Moreover, the relative gene expression of collagen type 2 and aggrecan was also increased (600-fold and 2-fold, respectively). Regarding osteogenic differentiation assay, relative gene expression for osteopontin increased for 3D- (150-fold) vs 2D-expanded cells. However, no difference was observed between 2D and 3D expansion for the adipogenic differentiation assay. DISCUSSION & CONCLUSIONS: The present study shows that 3D expansion of NP cells better preserves the progenitor's cells population and increases the chondrogenic and osteogenic differentiation potential compared to 2D expansion. This project not only has a scientific impact by evaluating the role of native physiological niches on the functionality of NP progenitors but could also lead to an innovative clinical approach with cell therapy for IVD regeneration and repair. Acknowledgments: Financial support was received from iPSpine H2020 project #825925

Gene expression variation between distinct areas of breast cancer measured from paraffin-embedded tissue cores

BACKGROUND: Diagnosis and prognosis in breast cancer are mainly based on histology and immunohistochemistry of formalin-fixed, paraffin-embedded (FFPE) material. Recently, gene expression analysis was shown to elucidate the biological variance between tumors and molecular markers were identified that led to new classification systems that provided better prognostic and predictive parameters. Archived FFPE samples represent an ideal source of tissue for translational research, as millions of tissue blocks exist from routine diagnostics and from clinical studies. These should be exploited to provide clinicians with more accurate prognostic and predictive information. Unfortunately, RNA derived from FFPE material is partially degraded and chemically modified and reliable gene expression measurement has only become successful after implementing novel and optimized procedures for RNA isolation, demodification and detection. METHODS: In this study we used tissue cylinders as known from the construction of tissue microarrays. RNA was isolated with a robust protocol recently developed for RNA derived from FFPE material. Gene expression was measured by quantitative reverse transcription PCR. RESULTS: Sixteen tissue blocks from 7 patients diagnosed with multiple histological subtypes of breast cancer were available for this study. After verification of appropriate localization, sufficient RNA yield and quality, 30 tissue cores were available for gene expression measurement on TaqMan(R) Low Density Arrays (16 invasive ductal carcinoma (IDC), 8 ductal carcinoma in situ (DCIS) and 6 normal tissue), and 14 tissue cores were lost. Gene expression values were used to calculate scores representing the proliferation status (PRO), the estrogen receptor status and the HER2 status. The PRO scores measured from entire sections were similar to PRO scores determined from IDC tissue cores. Scores determined from normal tissue cores consistently revealed lower PRO scores than cores derived from IDC or DCIS of the same block or from different blocks of the same patient. CONCLUSION: We have developed optimized protocols for RNA isolation from histologically distinct areas. RNA prepared from FFPE tissue cores is suitable for gene expression measurement by quantitative PCR. Distinct molecular scores could be determined from different cores of the same tumor specimen

Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue

The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months

Expression profiling with RNA from formalin-fixed, paraffin-embedded material

<p>Abstract</p> <p>Background</p> <p>Molecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge.</p> <p>Results</p> <p>We present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97.</p> <p>Conclusion</p> <p>We developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor.</p

Exploring a novel spheroid 3D cell culture system for Tie2+ nucleus pulposus cells of the intervertebral disc

Introduction Low back pain (LBP) is a big problem in our aging society. LBP impacts the patients quality of life and places an immense burden on the healthcare system worldwide [1]. Degeneration of the intervertebral disc (IVD) is one of the common causes of LBP. Recently, nucleus pulposus (NP) progenitor cells (NPPC) were discovered, which are positive for Angiopoietin-1 receptor (aka. Tie2+/CD202b) [2-5]. These NPPCs are a promising cell source for IVD regeneration and may spark the development of novel cell therapies. It is known, that these NPPCs are pretty rare (2-10% of all IVD cells) in human IVDs and diminish in number with increasing age [6]. The low numbers of sorted primary NPPCs necessitates an expansion of the NPPCs. It has been demonstrated, that 3D culture is superior to classic 2D culture to maintain the pluripotent phenotype of the NPPCs [3]. The goal of this research is to test the expansion and culture of bovine NPPCs cells in a novel spheroid plate. Material and Methods NP tissue was isolated from 5-6 discs form 4 bovine bovine tails (aged 10-13 months). NP cells were isolated by using a mild two step digestion protocol. Then, the primary NP cells were stained with Tie2 Antibody conjugated with Alexa 488 and DAPI for FACS. Sorted NP cells were expanded for 2 weeks under hypoxia (5% O2 ) and were supplemented with 2.5ng/ml FGF2 until 80% confluency. Tie2+ and Tie2- cells were then seeded in the functionalized and in the 2D control wells of the spheroid plate SP5D of Kugelmeier Ltd (see fig. 1). The cells were cultured for 2 weeks under hypoxia and FGF2. Colony forming unit-assay was performed at day 0. Cell activity, DNA/GAG content and gene expression of selected genes was measured on day 1, 8, 15. Results The DNA content of the Tie2+ and – spheroids (see fig. 2) increased by a factor of 1.7 and 3. The cells in the 2D plastic control wells grew faster and increased by factor of 11 and respectively 18. Figure 1: Spheroids of bovine NP Tie2+ cells stained with Calcein AM and Ethidium homodimer in the SP5D spheroid plate by Kugelmeier, 400 cells/microwell, 1d 6h after seeding Figure 2: Absolut DNA content/well, either 750 spheroids or 2D control in 24well plate, Tie2 + and Tie2-, cultivated under hypoxia (2% O2 ) and supplemented with FGF2 (N=1, n=3), means ± SD Conclusion The obtained results of the pilot show that sorted bovine NP cells proliferated in all culture conditions. There seems to be a big difference in the cell proliferation rate depending on the chosen culture system (3D vs 2D). However, more biological replicates are needed for further evaluation of the results. References 1. Spencer et al., Lancet 392:1789-858, 2018 2. Bischof et al., Applied Sciences 11: 7144, 2021 3. Guerrero et al., JOR SPINE 4:e1131, 2020. 4. Sako et al., Applied Sciences 11, 2021 5. Zhang, et al., Int. J. Mol. Sci 21: 9423, 2020 6. Sakai, et al., Nat Commun 3:1264, 2012 Acknowledgements This study is supported by an SNF Bridge – Discovery Grant (https://data.snf.ch/grants/grant/211510). FACSLab and Life cell imaging (LCI) of the DBMR, University of Bern are gratefully acknowledged

Trilineage Potency of Human Nucleus Pulposus Cells before and after Cryo-Preservation

INTRODUCTION:Low back pain (LBP) is a major cause of disability in many countries, affecting more than half a billion people worldwide. A promising and future-oriented approach to treat LBP is cell therapy using stem or progenitor cells. Over the last decade, cells positive for Tie2 and mesenchymal stromal cell markers have been found within the nucleus pulposus (NP) of human intervertebral discs (IVD). However, little is known about the effect of expansion and cryo-preservation on here called “heterogenic” human NP cells (hNPCs) and their stemness in a context of cell therapy for regeneration of the IVD. Therefore, the aim of our study was to expand hNPCs whilst investigating their differentiation potential before and after cryo-preservation and to find and optimal approach to cryo-preserve them. METHODS:HNPCs from three human trauma patients (32, 55 and 69 years old) undergoing spinal surgery were isolated with a mild two-step digestion protocol. After subsequent expansion until complete confluency, hNPCs were separated and then differentiated into osteogenic, adipogenic or chondrogenic lineages for 21 days or were cryo-preserved for one week at -150°C with five cryo-preservation media (90% fetal bovine serum and 10% dimethyl sulfoxide (DMSO); 90% low glucose medium + 10% DMSO and three commercially available media) to compare their effect on the cell’s viability and differentiation potential. Cell viability was determined with trypan blue and by cytometry employing propidium iodide. The differentiation potential was assessed using histological analysis and qPCR. RESULTS:HNPCs cultured in osteogenic medium showed a significant (p<0.01) higher expression of calcium deposits (up to 11-fold) vs. controls, indicating osteogenic differentiation. Furthermore, evidence for adipogenic and chondrogenic differentiation was observed using histological analysis and determining genes typical for chondrogenic and adipogenic lineages like collagen type 2 (up to 350-fold) or adiponectin (up to 3’700-fold). In addition, most hNPCs maintained their differentiation potential, even after cryo-preservation and independent of the cryo-preservation medium used. The hNPCs’ cell viability after storing for one week at -150°C was very similar for all conditions (~85% cell viability). DISCUSSION & CONCLUSIONS:The study showed heterogenic hNPCs have trilineage potential and as such possess stem cell characteristics. Therefore, they can potentially be used for future clinical trials concerning cell therapy for IVD regeneration. Furthermore, commercially available cryo-preservation media seem to perform just as well as homemade media in terms of cell viability and maintaining hNPCs differentiation potential

Mimicking a catabolic phenotype of intervertebral disc cells in 3D alginate culture

Introduction: Intervertebral Disc Degeneration (IDD) is a multifactorial pathological process often associated with chronic back pain. In the last decades a wide range of cytokines such as TNF-α or IL1-β have been applied in vitro to IVD cells to induce a catabolic phenotype to investigate the degenerative cascade. Hence, the aim of this study was to explore relevant catabolic phenotypes in-vitro in bovine intervertebral disc cells using 1.2% alginate beads as biological support after TNF