Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species

Background Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin‐1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2+ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation. Purpose Here, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence‐activated cell sorting‐based methodology to sort and analyze Tie2+ NPCs. Methods We present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2+ NPCs from the IVD cell population during the isolation process. A cross‐species phylogenetic analysis of Tie2 across species is presented. Results Our protocols are efficient towards labeling and isolation of Tie2+ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species. Conclusions Current identification of Tie2+ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2+ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research.

CD24. 3 With further aging and degeneration, the active NPCs dedifferentiate and their numbers decrease, altering the extracellular matrix (ECM) composition of the NP; changing from a proteoglycans and collagen type II-rich gelatinous tissue to a fibrous collagen type I-rich structure ( Figure 1C,D). 4 The reduced ECM quality results in decreased IVD water retention, tissue flexibility, and mechanical loading capacity along the spine. [5][6][7] Expression of Tie2/TEK receptor tyrosine kinase (Tie2; also known as CD202) was identified as a marker of local NP progenitor cell population by Sakai et al 8 in human and murine NPs. These Tie2 + NP progenitor cells were characterized by assessment of the total isolated NPC population in methylcellulose semisolid medium, commonly used in colony forming assays (CFA) for endothelial and hematopoietic progenitor cells. [8][9][10] Two distinct colonies developed, that is, (1) fibroblastic colony forming units (CFU-F) and (2) spherical colony forming units (CFU-S), named based on their morphology ( Figure 2). High collagen type II and aggrecan protein expression was exclusively observed within CFU-S. Subsequently, NPCs surface marker was correlated to CFU-S forming capacity, and strong relationship was observed with Tie2 expression. NPCs were sorted based on Tie2 immunoreactivity and subsequently reassessed by CFA. This resulted in a high frequency of CFU-S in Tie2 + populations but not for Tie2 − populations. Notably, Tie2 + cells were identified as the precursor that further differentiated and started to express other surface markers, including GD2 (disialoganglioside 2) and CD24. 8 Moreover, Tie2 + cells showed the ability of cell renewal, which is lost with decrease of Tie2 expression. 8 Next, Tie2 + NPCs were assessed on marker expression of macrophage, endothelial cell or pericyte markers (ie, CD11c, CD14, CD31, CD34, CD45, CD144, CD146, and Von Willebrand Factor) to exclude potential contamination, which resulted in no detected immunoreactivity. Also, direct immunostaining against Tie2 in IVD sections revealed distinct Tie2 expression in human and canine NPs ( Figure S1, Supporting Information). To further investigate the progenitor cell characteristics of the Tie2 + NPCs, differentiation towards osteogenic, chondrogenic, and adipogenic lineages was successfully performed. Moreover, we observed that Tie2 positivity in the NPC population rapidly decreased with progression of IVD degeneration and aging in humans 8 ( Figure 3A) and mice. 11 Li et al 12 later demonstrated that despite the relatively low Tie2 + NPCs numbers obtainable from degenerated IVD from patients with degenerative IVD disease, proliferation rate, lineage differentiation potency, and regenerative capacity was maintained. Interestingly, these Tie2 + NPCs had a superior differentiation capacity towards the chondrogenic lineage compared with bone marrow-derived mesenchymal stromal cells from the same patients. 12 Other studies demonstrated that transplanted Tie2 + NPCs could be differentiated towards Schwan-like cells resulting in improved functional recovery in murine sciatic peripheral nerves, again substantiating the progenitor-like nature. 13 Nevertheless, Tie2 + NPCs application remains limited due to the rapid reduction in ratio of Tie2 + NPCs with in vitro expansion ( Figure 3B). 8,14,15 In particular, Tekari et al 15 showed that Tie2 expression in bovine NPCs decreased from approximately 8% to less than 1% after 2.3 population doublings. Tie2 + preservation could, however, be augmented under hypoxic conditions, by supplementation of fibroblast growth factor 2 (FGF2) or synergistic FGF2 and hypoxic conditions ( Figure 3C). 16 demonstrated a lack of Tie2 expression during the development of the fetal notochord up to 18 weeks postconception, indicating that Tie2 might not play a role during development. Nonetheless, after fetal IVD explantation, isolation, and culture we were able to detect Tie2 + NPCs from both human and canine fetal tissue ( Figure S1). Cumulatively, these data suggest a role for Tie2 + NPCs as an early-stage response to disruption in the environment to restore homeostasis of the IVD. 8 Cell-based strategies for the treatment of chronic back and neck pain due to IVD degeneration have gained significant momentum, 17 but an optimal cell source remains elusive. Considering their clonal ability for multilineage differentiation potential comparable or even superior to mesenchymal stromal cells, 12 the Tie2 + NPCs can be considered a promising cell transplantation candidate. Tie2 + NPCs isolation not only provides an opportunity to study IVD homeostasis and the pathogenesis of IVD degeneration, but it also enables studying the (regenerative) effects of Tie2 + NPCs upon other cell residing in the NP, for example, NPCs from degenerated IVDs. Tie2 + NPCs or their (recombinantly made) bioactive secreted factors could eventually be applied intradiscally to support IVD regeneration. Thus, there is a need for a methodology to analyze and isolate Tie2 + NPCs properly and to enable their application for research and therapeutic purposes.

Rodrigues-Pinto et al
To allow for more reproducible Tie2 + NPCs isolation, we here describe extensively an optimized methodology formulated by a multidisciplinary group of experts with experience in Tie2 + NPCs research for the harvest, identification, isolation, characterization, and subsequent multiplication of Tie2 + NPCs from relevant species commonly applied in IVD research, that is, in human, canine, bovine, and murine origin. We first provide a tissue selection and harvest method protocol, including the pitfalls that could limit Tie2 + NPCs yield. Lastly, we provide a complete overview of appropriate gating for FCM and FACS analysis to enhance isolation of the Tie2 + NPC population and permit CFU-S formation.

| Experimental design
The Tie2 + NPCs isolation protocol is divided into the following sec-  The intervertebral disc and degenerative disc disease. Schematic and a hematoxylin/eosin-stained histological sections of a healthy (A, B) and degenerated (C, D) canine intervertebral disc. (E) Human lumbar intervertebral disc obtained postmortem. Appropriate tissue selection for Tie2 fishing is indicated by dashed black lines, while degenerated or AF tissue areas are indicated by dashed red lines. (F) Human intervertebral disc (IVD)-derived tissues during discectomy are selected based on morphology, color and stiffness. Only gelatinous, white, and transparent tissue should be used for Tie2 fishing. Yellow and stiff tissue should be disregarded in order to enhance Tie2 detection

| Tissue selection and NPC isolation
The age of the human donor is a crucial indicator of the percentage of Tie2 + cells ( Figure 3A). Moreover, age also diminishes the number of viable NPCs ( Figure 3B), further complicating Tie2 + NPCs isolation and analysis. Finally, IVDs afflicted by certain pathologies, such as IVD degeneration, have been shown to correlate with a reduced percentage of Tie2 + NPCs. 8 Thus, for optimal results, young, minimally degenerated IVD samples are applied. For murine 11 and canine donors, age is also an important negative determinant.
For all species, the tissue is harvested within 1 to 2 days postmortem and cell isolation is performed within 24 hours to preserve Tie2 + cells and overall cell viability. IVD tissue removed during surgery is kept in serum free medium and stored in a container placed on ice at the day of explantation to preserve cell viability. Alternatively, the IVD tissue can be stored in wet gauzes within a closed container to FIGURE 2 Colony forming units from assessed species. Nucleus pulposus progenitor cells from different species sorted on Tie2 expression were cultured in semi-solid methylcellulose medium. Fibroblastic colony forming units (CFU-F) and spherical colony forming units (CFU-S) emanate as 2 distinguishable colony types. Scale bar represents 50 μm FIGURE 3 Nucleus pulposus cell viability and Tie2 expression negatively correlates with age. Flow cytometric analysis of human primary nucleus pulposus cells (NPCs) isolated using enzymatic digestion. (A) Percentage of Tie2 + NPCs related to age shows a steep decline after 25 years of age (R 2 = 0.9489). (B) Gating by PI threshold reveals a strong decline in percentage of viable NPCs isolated by enzymatic intervertebral disc (IVD) digestion with age. (C) Tie2 + NPCs, isolated by flow-activated cell sorting for Tie2. Primary refers to NPC that were sorted for negative (Tie2 − ) and positive (Tie2 + ) Tie2 cell populations and were included as controls. Tie2 + NPC were expanded in vitro for 7 days (expanded) in αMEM +10% (v/v) FBS in normoxic culture conditions either alone (−), or supplemented with either 10 ng/mL transforming growth factor beta (TGF-β) or 100 ng/mL fibroblast growth factor 2 (FGF2) or subjected to hypoxic culture conditions. Fold increase in gene (mRNA) and protein (protein) expression levels were determined in Tie2 + NPCs by qPCR and flow cytometry, respectively in primary NPCs (Tie2 − and Tie2 + after sorting) and in expanded Tie2 + NPCs. mRNA and protein expression levels in primary Tie2 − NPC were set at 1. *P < .05 and # P < .005 as compared to normoxic expanded Tie2 + NPCs. (D) the capacity of sorted Tie2 + NPCs to form spherical colony forming units (CFU-S), assessed using methylcellulose semi-solid culture medium for 10 days, decreases with age ensure humidified conditions at 4 C. Prior to cell isolation, it is crucial to establish aseptic working conditions. Within this protocol, solutions are supplemented with penicillin and streptomycin, however additional antibiotics can be considered for tissues derived from less sterile sources, in particular for cadaveric-derived tissues and primary cultures.
Isolation of NP tissue from the IVD requires careful separation from the surrounding tissue types. In case of full IVD explantation, a cut through the AF is made at the height of the EP, and dismembering one vertebra from the IVD. Thereafter, NP tissue is excised by macroscopic examination. For smaller samples (eg, murine or fetal-stillborn human and canine tissue), an inverted microscope or binocular glasses are highly recommended. In case of partial IVD explantation, for example, after discectomy, microscopic examination is required to distinguish and separate the gelatinous NP tissue from the fibrous AF. Highly degenerated, fibrotic, or calcified NP tissue segments are avoided. After extraction, large NP samples are minced in approximately 0.3 cm 3 pieces, while the cells are obtained by enzymatic digestion of the surrounding ECM. It is furthermore recommended to apply specific enzymes and concentrations per species, as the NP tissue size and composition differs considerably between species. 18 The isolated NPCs can either be analyzed directly (FCM: step 28-53, FACS: step 54-69), or expanded first (step 21-27).

| NPC expansion culture
NPC expansion can increase cell numbers for FCM or FACS analysis, which is particularly useful for small tissue sizes or if FACS/FCM analysis is not available immediately after NP digestion. The expansion is ideally performed in αMEM with 10% (v/v) FBS under hypoxic conditions (5% O 2 ), to augment maintenance of the Tie2 + NPCs phenotype. 12 Supplementation of 100 ng/mL FGF2, limited culture time and cell passaging can limit the loss of Tie2 expression ( Figure 3C). 15 For murine specifically, it is highly recommended to expand primary isolated cells to induce Tie2 expression.

| FCM and FACS data acquisition and analysis
For this methodology report, FCM and FACS assessment are   Figure 3B) and low Tie2 expression ( Figure 3A), making it difficult to , indicates successful FCM analysis; ☓, indicates lack of reactivity; ND, indicates that the antibodies reactivity has not been determined towards this species.
• EDTA•2Na (DOJINDO,   should be reduced to a minimum in these samples.  instead of 60 to 120 minutes.
14. Decant the cell suspension onto a 40-μm cell strainer on a 50-mL tube, removing undigested pieces of tissue by filtration.
Discard the strainer.  Table 3 for troubleshooting solutions.)  Table 3   Lower the enzyme concentration and/or enzymatic digestion time.
In very gelatinous NP tissue, the pronase step could be reduced/ omitted.

No/very low number of viable cells present after cryopreservation
Cell damage by cryopreservation (in particular for fetal cells) Culture cells for about 7 days and analyze or cryopreserve thereafter.

NPC population cannot be recognized
Cell suspension contains high numbers of dead cells, tissue debris, and non-NPC.
To remove erythrocytes and tissue and cell debris, lymphoprep (StemCell Technology, Cat. No. 07851) can be applied.
Limited NPCs are present Set the FSC-H and SSC-H gate over a larger region that captures 500 000 cells and narrow the gate later during the procedures to further specify the correct NPC population.
Release of antibody by harsh and repeated washing (in particular observed for fetal samples) Limit the mechanical force generated during the washing step with FACS buffer, which might separate the antibody from its antigen, by gently adding the solutions via the inner wall of the tube in a slightly tilted position.
Reduce the number of washing repetitions during the staining procedures 52 Tie2 positive cells cannot be detected The examined tissue is derived from aged, diseased, or degenerated tissue.
Do not digest tissue, but allow cells to be cultured inside their tissue sections prior to enzymatic digestion. To remove erythrocytes and tissue and cell debris, lymphoprep (StemCell Technology, Cat. No. 07851) can be applied.
Limited NPCs are present Set the FSC-H and SSC-H gate over a larger region that captures 500 000 cells and narrow the gate later during the procedures to further specify the correct NPC population.

Release of antibody by harsh and repeated washing
Limit the mechanical force generated during the washing step with FACS buffer, which might separate the antibody from its antigen, by gently adding the solutions via the inner wall of the tube in a slightly tilted position.
Reduce the number of washing repetitions during the staining procedures Critical: Vacuolated notochordal cells will adhere to the plate either as single cells or in clusters and will gradually lose their vacuolated phenotype over the period of 7 to 10 days. 20 22. Upon 70% to 80% confluency, aspirate and discard the culture medium and rinse the cell layer with 10 mL of PBS. Subsequently, discard the PBS and cover the monolayer culture with   Critical: Assure that when applying a secondary antibody, the antibody is not reactive to the species of the tissue material, and is reactive to species from which the primary antibody was derived.
34. Leave on ice, kept from light, for 30 minutes.  Table 3 for troubleshooting solutions.  Step wise overview of FCM gating. Schematic overview of procedural steps for appropriate gating of Tie2 + NPCs. Percentages indicate the positivity for Tie2 in the particular gate

TIMING
Step 1 to 20 Nucleus pulposus cell isolation: 5 to 10 hours Step 21 to 27 Expansion of nucleus pulposus cells (optional):

days
Step 28 to 37 Antibody staining: 1 to 2 hours Step 38 to 45 Data collection by FACS: 2 hours Step 46 to 53 Data analysis and Tie2 detection: 2 hours Step 54 to 69 Sorting of Tie2 + cells: 3 hours Step 70 to 80 Colony-forming assay: 7 to 10 days

| RESULTS
Tie2 expression rapidly reduces in NP tissue obtained from patients above the age of 25, while NPs from patients older than 30 years of age demonstrate less than 10% Tie2 expression ( Figure 3A). Similarly, Tie2 expression considerably decreases with IVD degeneration, demonstrating an average positivity of approximately 14% for grade III IVDs and less than 2.5% positivity for grade IV IVDs. 8 As the IVD tissue applied for research purposes is commonly explanted during surgery as part of a treatment of IVD degeneration or scoliosis, or as postmortem explantation, it is most likely to be prone to low Tie2 + rates. To successfully detect and isolate sufficient Tie2 + NPCs from human NP tissue, it is crucial to select appropriate tissue sources and set-up isolation procedures that minimize the loss of Tie2 expression.
Interestingly, the percentage of Tie2 + cells differs considerably between fetal human donors ( Figure S1), and is generally lower than in young adult individuals. FACS analysis showed only 0% to 17% Tie2 positivity for 22 to 24-week-old human fetuses, but these results might have been influenced by cell multiplication, since NPCs of fetal/ stillborn individuals need to be expanded to yield >10 3 cells for FCM or FACS analysis, negatively affecting Tie2 expression. Additionally, it was established that the number of washing steps significantly reduced Tie2 positivity in these specific fetal samples (data not shown). We hypothesize that the mechanical force generated during the washing step with excessive FACS buffer separates the antibody from its antigen, suggesting a weak affinity to the fetal Tie2 membrane receptor.

| DISCUSSION
The percentage of Tie2 + cells in stillborn canine NP tissue also varies considerably between donors ( Figure S1) and was assessed in the range of 12% to 43% (data not shown), as observed with FACS analysis. Again, these results might have been influenced by cell passaging and cryopreservation, since the NPCs needed to be expanded to obtain sufficient cell numbers for FCM or FACS analysis. Since cell passaging negatively affects Tie2 positivity, the percentage of Tie2 + NPCs might have been higher in freshly isolated stillborn canine NPCs. Similar to humans, Tie2 positivity rapidly decreases with age in canines, since NP tissue from 1 until 5-year-old canine donors contains only 0% to 1% Tie2 + NPCs. Therefore, it is crucial to select very young (<1-year-old) canine donors for Tie2-related research purposes.
In bovine NP tissue, the number of obtained Tie2 + cells after digestion of the NP tissue constitutes approximately 5% to 12% from the entire NPC population. No important variation in the number of Tie2 + NPCs after isolation was detected, since the bovine coccygeal discs were homogenous and obtained from 8 to 12 months old animals from the local abattoir. It should be noted that only healthy discs were harvested with no sign of degeneration or trauma.
Murine samples also demonstrate an age-related Tie2 + ratio with a relatively low expression observed from 6 months of age. 11 Additionally, for murine samples in particular, primary NPCs show low Tie2 + ratio.