The proteome signature of cord blood plasma with high hematopoietic stem and progenitor cell count.

Hematopoietic stem and progenitor cells (HSPC) from umbilical cord blood (UCB) are used for transplantation to treat blood disorders. Methods to estimate the HSPC count in umbilical cord blood, and thereby identify high-value blood units, are time-consuming and costly. Recent studies indicate that the UCB plasma protein composition relates to the HSPC count. We compared the plasma proteome of UCB with high vs low HSPC cell count (>115 × 106 vs < 51 × 106 CD34+ cells l-1) by using a combination of global untargeted MS quantitative proteomics and targeted proximity extension assay (PEA) proteomics. For the MS platform, 96 proteins differed significantly between the CD34+ groups, and out of these, 44 proteins showed more than a two-fold difference. Seven pathways were enriched in high CD34+ samples, including pathways relating to platelets, coagulation, and lipid transport. For the PEA platform, 61 proteins were differentially abundant, and among these 7 proteins showed more than a two-fold difference between groups. In the PEA data, a high CD34+ cell count was associated with a protein hub with functions in platelet degranulation. We conclude that the HSPC count is related to the UCB plasma proteome, but that further studies are needed to discern if these findings reflect causal relationships.

Terminal complement complex formation is associated with intervertebral disc degeneration.

PURPOSE: The complement system is a crucial part of innate immunity. Recent work demonstrated an unexpected contribution to tissue homeostasis and degeneration. This study investigated for the first time, in human disc tissues, the deposition profile of the complement activation product terminal complement complex (TCC), an inflammatory trigger and inducer of cell lysis, and its inhibitor CD59, and their correlation with the degree of disc degeneration (DD). METHODS: Disc biopsies were collected from patients diagnosed with DD (n = 39, age 63 ± 12) and adolescent idiopathic scoliosis (AIS, n = 10, age 17 ± 4) and compared with discs from healthy Young (n = 11, age 7 ± 7) and Elder (n = 10, age 65 ± 15) donors. Immunohistochemical detection of TCC and CD59 in nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) was correlated with age, Pfirrmann grade and Modic changes. RESULTS: Higher percentage of TCC+ cells was detected in the NP and EP of DD compared to Elder (P < 0.05), and in the EP of Young versus Elder (P < 0.001). In DD, TCC deposition was positively correlated with Pfirrmann grade, but not with Modic changes, whereas for Young donors, a negative correlation was found with age, indicating TCC's involvement not only in DD, but also in early stages of skeletal development. Higher CD59 positivity was found in AIS and DD groups compared to Young (P < 0.05), and it was negatively correlated with the age of the patients. CONCLUSION: TCC deposition positively correlated with the degree of disc degeneration. A functional relevance of TCC may exist in DD, representing a potential target for new therapeutics.

Intradiscal Injection of Iron-Labeled Autologous Mesenchymal Stromal Cells in Patients With Chronic Low Back Pain: A Feasibility Study With 2 Years Follow-Up.

PURPOSE: Degeneration of the intervertebral disc is considered to be central in pain pathogenesis in patients suffering from chronic low back pain (LBP). In recent years, the injection of mesenchymal stromal cells (MSCs) into the disc to arrest or reverse the degenerative process has been proposed as an alternative therapy. The aim of the present study was to investigate the feasibility of using iron-labeled MSCs for intradiscal injection in patients with long-standing LBP. METHODS: Ten patients (7 men, 3 women, mean age 40 years, range 26-53) with chronic LBP and confirmed disc degeneration on magnetic resonance imaging (MRI) were recruited from the waiting list for planned surgery. Injection of autologous, expanded, and iron-labeled bone marrow-derived MSCs (BM-MSCs) into 1 or 2 disc levels was undertaken. Follow-up consisted of monitoring of adverse events, regular MRI examinations, and collection of patient-reported outcome measures (PROMs) for a minimum of 2 years. RESULTS: No complications could be detected, neither clinically nor on MRI. No statistically significant improvement was seen for PROMs on a group level up to 2 years postinjection. Three of 10 patients opted to proceed with the initially planned surgery within the first year and 2 more within 3 years postinjection. CONCLUSION: Results from this pilot cohort study show that injection of autologous expanded iron-labeled BM-MSCs is a safe procedure, in accordance with the existing body of evidence. The clinical result warrants further larger studies. LEVEL OF EVIDENCE: 4 for therapeutic studies.

BMP-3 Promotes Matrix Production in Co-cultured Stem Cells and Disc Cells from Low Back Pain Patients.

Low back pain is one of the most common disorders and believed to be due to intervertebral disc degeneration. Transplantation of human mesenchymal stem cells (hMSCs) is suggested as potential treatment option. Bone morphogenetic growth factor 3 (BMP-3) promotes chondrogenesis and is proven effective in enhancing chondrogenesis in hMSCs pretreated with interleukin-1 beta (IL-1ß) in hydrogel model. Three-dimensional co-cultures of hMSCs and disc cells (DCs) have previously been demonstrated to result in increased proteoglycan production. The aim was to study the effects of BMP-3 on hMSCs, DCs, as well as hMSCs and DCs in co-culture in a pellet system, both as single treatment and after pretreatment of IL-1ß. Cell pellet cultures with hMSCs, DCs, and co-culture (1:1 ratio) were performed and stimulated with BMP-3 at 1 or 10 ng/mL concentrations. For pretreatment (PRE-T), cell pellets were first stimulated with IL-1ß, for 24 h, and then BMP-3. The pellets were harvested on day 7, 14, and 28. Results demonstrated that BMP-3 stimulation at 10 ng/mL promoted cell viability, proteoglycan accumulation, as well as chondrogenesis in all pellet groups compared to 1 ng/mL. Cellular proliferation and chondrogenic differentiation of hMSCs were best promoted by PRE-T at 10 ng/mL, whereas BMP-3 best enhanced chondrogenesis in DC and co-culture pellets at the same concentration. Impact Statement Current therapies for low back pain include pain modulation and surgery, which do not tackle the underlying cellular mechanisms of the degenerated intervertebral discs (IVDs). To develop an understanding of the degeneration process and to further reverse its course, the effects of growth factor and cytokine on the native cells of the IVDs were investigated, revealing the potency of bone morphogenetic growth factor 3 on disc cells (DCs) and combined culture of mesenchymal stem cells and DCs. These results may impact future strategies in development of cell therapies that could directly influence the IVD degeneration process, which might alter the treatment models of today.

Pathological Effects of Cortisol on Intervertebral Disc Cells and Mesenchymal Stem Cells from Lower Back Pain Patients.

In western countries, lower back pain (LBP) is one of the most common disorders, experienced by more than 80% of the population. Chronic LBP due to disc degeneration has been linked to ongoing inflammatory processes in the disc and endplates. Pain effects the body in different ways, inducing a general stress response in which the body responds by releasing the stress hormone cortisol. Little is known about the impact of pain-induced stress on the progression of disc degeneration. Thus, the effects of cortisol on disc cells (DCs) and human mesenchymal stem cells (hMSCs) were explored in vitro with the objective of investigating the repercussions of cortisol on these cell types involved in de- and regenerative mechanisms of the disc. DC and hMSC pellet cultures were exposed to cortisol at two concentrations (150 and 300 ng/mL) for 28 days to simulate pain-induced stress. Cell viability, histological staining, and GAG DNA, along with apo-ptotic assays were conducted. Detection of OCT4, SOX9, IL-1R, and CXCR2 expressions was performed by immunohistochemistry. With cortisol treatment, restricted cell proliferation and less GAG production in both DCs and hMSCs were observed. Suppression of the differentiation and immunomodulatory efficacy of hMSCs was also detected. Moreover, elevated expressions of IL-1R and CXCR2 were detected in both cell types. To conclude, constant exposure to cortisol even at a physiological level enhanced pathological cellular processes in both DCs and hMSCs, which further jeopardized chondrogenesis. This suggests that cortisol resulting from pain-induced stress is a contributing component of intervertebral disc degeneration and may negatively affect regenerative attempts of the disc.

Human Mesenchymal Stem Cells Pretreated with Interleukin-1ß and Stimulated with Bone Morphogenetic Growth Factor-3 Enhance Chondrogenesis.

Low back pain is one of the most common ailments in western countries afflicting more than 80% of the population, and the main cause is considered to be degeneration of intervertebral discs. Interleukin-1ß (IL-1ß) is a vital inflammatory cytokine found in abundance in degenerated disc environment, whereas bone morphogenetic growth factor-3 (BMP-3) is believed to promote chondrogenesis through transforming growth factor-beta (TGF-ß) pathway. The aim was to study the effects of BMP-3, IL-1ß, and combination (pretreatment with IL-1ß) on human mesenchymal stem cells (hMSCs) encapsulated in PuraMatrix™ hydrogel (Phg) especially in the absence of TGF-ß in order to investigate the proliferation and differentiation ability of hMSCs over 28-day period. One hundred microliters of hMSCs' cell suspension was encapsulated between two layers of 100 µL hydrogels forming a sandwich-like structure. The encapsulated hMSCs were cultured in two sets of media, chondrogenic (C) and nonchondrogenic (nC) media, along with addition of BMP-3 (10 ng/mL) and IL-1ß (10 ng/mL). To study the combined effects of BMP-3 and IL-1ß, the encapsulated hMSCs were first pretreated with relevant media containing IL-1ß for 24 h, and then the media was replaced by media containing BMP-3 for the remaining experimental time period. IL-1ß pretreatment was carried out in both C and nC media. The samples were collected at day 7, 14, and 28. Proliferation and differentiation of hMSCs into chondrocyte-like cells were observed in all samples. Proteoglycan accumulation was observed in pretreatment samples in C media. The protein and gene expression of Sox-9 and COL2A1, respectively, showed the occurrence of chondrogenesis in all samples. High cell viability, proliferation, and differentiation were achieved in this in vitro model confirming that BMP-3 alone in the absence of TGF-ß could drive hMSCs into chondrogenic lineage. Pretreatment with IL-1ß followed by BMP-3 stimulation resulted in high proteoglycan accumulation compared to stimulation with growth factors or cytokine alone. This suggests that pretreatment with a pro-inflammatory cytokine before driving them into a chondrogenic lineage might be of importance also in vivo.

Iron sucrose-labeled human mesenchymal stem cells: in vitro multilineage capability and in vivo traceability in a lapine xenotransplantation model.

For evaluation of cell therapy applications, it is of interest to be able to trace and observe cellular distribution of the transplanted cells. The aim with the study was to examine viability, traceability, and multilineage capability of iron sucrose-labeled mesenchymal stem cells (MSCs) after transplantation into lapine intervertebral discs (IVDs). MSCs were collected from three human donors, age 31-50 years, and IVDs from 12 rabbits, age 3 months. MSCs were isolated from the bone marrow and cultured using standard protocols. Iron sucrose labeling of MSCs was performed in Dulbecco's Modified Eagle's Medium-low glucose with Venofer(®). The iron sucrose-labeled MSCs were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. Results were evaluated using Oil red, von Kossa, Alcian blue, and collagen II (immunohistochemistry). For the animal experiments, iron sucrose-labeled MSCs and nonlabeled MSCs were injected into lapine IVDs (LI-LIV level). After transplantation, at the time points of 1 and 3 months, IVDs were collected and cells were analyzed for cell viability (fluorescence-activated cell sorting). The lapine IVDs were collected and examined for presence of cells positive for iron deposits using Berliner blue staining. Differentiation of the iron sucrose-labeled MSCs into adipogenic (lipid droplets), osteogenic (calcium deposits), and chondrogenic lineage (proteoglycan/collagen II accumulation) (3/3 donors) was observed in vitro. After transplantation, the mean cell viability for iron-labeled MSCs/IVD cells was 99%, for nonlabeled MSCs/IVD cells was 95%, and for control IVD cells was 99% at a time point of 1 month. At a time point of 3 months, mean cell viability was 73% for iron sucrose-labeled MSCs/IVD cells, for nonlabeled MSCs/IVD cells was 77%, and for control IVD cells was 98%. At the time point of 1 month, cells positive for iron deposits were detected sparsely distributed in IVDs (tissue sections) in 4/4 animals and at the time point of 3 months in 4/4 animals. The results indicate that iron sucrose can be used as a cell tracer with a stable detection potential in tissues (histologies). This may be an important evaluation tool for understanding stem cell distribution/function after transplantation into degenerated cartilaginous tissues.

Cell viability and chondrogenic differentiation capability of human mesenchymal stem cells after iron labeling with iron sucrose.

For evaluation of cell therapy strategies using human mesenchymal stem cells (hMSCs), it is important to be able to trace transplanted cells and their distribution in tissues, for example, cartilage, over time. The aim of the study was to determine effects on cell viability, traceability, and chondrogenic differentiation of hMSCs after iron labeling with iron sucrose. hMSCs were collected (seven donors, 13-57 years) from patients undergoing spinal surgery. Two subsets of experiments were performed. (1) Iron labeling of hMSCs: 1 mg/mL of Venofer(®) (iron sucrose) was added (16 h) to cultures. hMSCs were examined for uptake of iron sucrose (Prussian blue staining) and cell viability (flow cytometry). (2) Iron-labeled hMSCs (passage 4) (n=4, pellet mass), 200,000 cells/tube, were cultured (DMEM-HG) with 10 ng/mL TGFß and compared with controls (from each donor). The pellets were harvested at days 7, 14, and 28. Real-time PCR, IHC, and histology were used to evaluate SOX9, ACAN, C6S, and COL2A1 expression. Mean number of cells containing iron deposits was 98.1% and mean cell viability was 92.7% (no significant difference compared with unlabeled control cells). Pellets containing iron-labeled cells expressed COL2A1 on protein level (all time points), in similar levels as controls, and glycosaminoglycan accumulation was observed in iron-labeled pellets (day 14 or day 28). Results were supported by the expression of chondrogenic genes SOX9, ACAN, and COL2A1. The results in vitro indicate that iron sucrose can be used as a cell tracer for evaluation of cellular distribution in vivo after transplantation of MSCs and thus contribute with important knowledge when exploring new treatment strategies for degenerated cartilaginous tissues.

Similar cellular migration patterns from niches in intervertebral disc and in knee-joint regions detected by in situ labeling: an experimental study in the New Zealand white rabbit.

INTRODUCTION: Potential stem cell niches (SNs) were recently reported in intervertebral discs (IVDs) and knee joints (KJs) in different mammals (located adjacent to the epiphyseal plate; EP). The aim here was to examine further possible cellular migration and migration directions of cells originating from niches possibly involved in regeneration of cartilaginous tissues in the IVD and in the KJ regions in adult mammals. METHODS: In total, 33 rabbits were used in studies A through C. A. IVD cells were sorted; fluorescence-activated cell sorting (FACS) by size (forward scatter; ≤ 10 µm or >10 µm or GDF5+ cells (anti-GDF5 antibody). Sorted cells, labeled with cell tracer (carboxyfluorescein-diacetate-succinimidyl ester; CDFA-SE) were applied on IVD explants in vitro. Migrating cells/distance was evaluated by fluorescence- and confocal-microscopy (FC). B. DNA labeling was performed with BrdU (oral administration). Animals were killed (14 to 56 days), KJs collected, and BrdU+ cells visualized with immunohistochemistry (IHC)/anti-BrdU antibody in SN and articular cartilage (AC). C. Cell tracer: (Fe-nanoparticles: Endorem) were injected into SNs of IVDs (LI-LV) and KJs (tibia). Animals were killed after 2 to 6 weeks. Fe-labeled cells were traced by ferric-iron staining (Prussian blue reaction; Mallory method). RESULTS: A. GDF5+ cells and ≤ 10-µm cells displayed the best migration capability in IVD explants. GDF5+ cells were detected at a tissue depth of 1,300 µm (16 days). B. BrdU+ cells were observed in early time points in niches of KJs, and at later time points in AC, indicating a gradual migration of cells. C. Fe+ cells were detected in IVDs; in annulus fibrosus (AF) in 11 of 12 animals and in nucleus pulposus (NP) in two of 12 animals. In AC (tibia), Fe+ cells were detected in six of 12 animals. In the potential migration route (PMR), from niches toward the IVD, Fe+ cells (three of 12 animals) and in PMR toward AC (KJs) (six of 12 animals) were detected. CONCLUSIONS: Results indicate similar cellular migration patterns in cartilage regions (IVD and KJs) with migration from stem cell niche areas into the mature cartilaginous tissues of both the KJs and the IVD. These findings of a cellular migration pattern in mature cartilage are of interest from tissue-repair and engineering perspectives.

The presence of local mesenchymal progenitor cells in human degenerated intervertebral discs and possibilities to influence these in vitro: a descriptive study in humans.

Low back pain is common and degenerated discs (DDs) are believed to be a major cause. In non-degenerated intervertebral discs (IVDs) presence of stem/progenitor cells was recently reported in different mammals (rabbit, rat, pig). Understanding processes of disc degeneration and regenerative mechanisms within DDs is important. The aim of the study was to examine the presence of local stem/progenitor cells in human DDs and if these cell populations could respond to paracrine stimulation in vitro. Tissue biopsies from the IVD region (L3-S1) were collected from 15 patients, age 34-69 years, undergoing surgery (spinal fusion) and mesenchymal stem cells (MSCs) (iliac crest) from 2 donors. Non-DD cells were collected from 1 donor (scoliosis) and chordoma tissue was obtained from (positive control, stem cell markers) 2 donors. The IVD biopsies were investigated for gene and protein expression of: OCT3/4, CD105, CD90, STRO-1, and NOTCH1. DD cell cultures (pellet mass) were performed with conditioned media from MSCs and non-degenerated IVD cells. Pellets were investigated after 7, 14, 28 days for the same stem cell markers as above. Gene expression of OCT3/4 and STRO-1 was detected in 13/15 patient samples, CD105 in 14/15 samples, and CD90 and NOTCH1 were detected 15/15 samples. Immunohistochemistry analysis supported findings on the protein level, in cells sparsely distributed in DDs tissues. DDs cell cultures displayed more undifferentiated appearance with increased expression of CD105, CD90, STRO-1, OCT3/4, NOTCH1, and JAGGED1, which was observed when cultured in conditioned cell culture media from MSCs compared to cell cultures cultured with conditioned media from non-DD cells. Expression of OCT3/4 (multipotency marker) and NOTCH1 (regulator of cell fate), MSC-markers, CD105, CD90, and STRO-1, indicate that primitive cell populations are present within DDs. Furthermore, the possibility to influence cells from DDs by paracrine signaling /soluble factors from MSCs and from nondegenerated IVD cells was observed in vitro indicating that repair processes within human DDs may be stimulated.