Systemic Transplantation of Adult Multipotent Stem Cells Functionally Rejuvenates Aged Articular Cartilage

Osteoarthritis (OA) is the most common and debilitating joint disease of advanced age and has no universally effective therapy. Here, we demonstrate that systemic transplantation of adult multipotent muscle-derived stem/progenitor cells (MDSPCs)—isolated from young mice—rejuvenates the knee articular cartilage (AC) of naturally aged mice. This intervention reduced expression of pro-inflammatory cytokines (Tnf and Il1a) and catabolic matrix-degrading proteinases (Mmp3 and Mmp13) in aged cartilage. Treatment with young MDSPCs also increased expression of pro-regenerative (Col2a1 and Acan) and prolongevity genes (Pot1b), including those associated with chondrocyte proliferation and differentiation, cartilage growth, and telomere protection. Indeed, the AC of MDSPC-treated mice exhibited reduced age-related histological pathologies. Importantly, the reduced mobility and arthritis-related gait dysfunctions of aged mice were also ameliorated by this treatment. Together, our findings demonstrate the rejuvenating effects of systemic transplantation of young MDSPCs on aging AC—at the molecular, tissue, and functional levels. This suggests that MDSPCs, or their secreted factors, may represent a novel therapy that can increase mobility and function in aged or OA patients.


MDSPC transplantation
All animal experiments were performed with the approval of the Northwestern University Institutional Animal Care and Use Committee. Female WT C57BL/6 mice 20 months of age were obtained from the National Institute of Aging (NIA). The mice were fed ad libitum a gammairradiated standard chow. Phosphate buffered saline (PBS) or MDSPCs suspended in PBS were transplanted via intraperitoneal (IP) administration into 22-month-old mice at 2 x 10 5 MDSPCs per gram body-weight. Mice were sacrificed two months following transplantation at 24 months of age; the right hindlimbs were harvested for histopathological analysis and the left knee joints were used for gene expression analysis.

RNA isolation and qPCR
To measure mRNA expression, total RNA was extracted from AC isolated from paraformaldehyde (PFA)-fixed knee joints using a RNeasy® mini kit for formalin-fixed, paraffin-embedded (FFPE) tissue (Qiagen) according to manufacturer's protocol. RNA quality was validated using an Eppendorf Bio-Spectrophotometer, and 100 ng of total RNA was reverse-transcribed according to the manufacture's protocol using iScript Advanced cDNA synthesis kit (Bio-Rad Laboratories). Pre-amplification of the primers was carried out according to the manufacturer's protocol using a Pre-amplification kit (Bio-Rad Laboratories). The pre-amplified cDNA was diluted and used for analysis of gene expression changes in 10 L reactions using SYBR green advanced master mix kit (Bio-Rad Laboratories) and the gene of interest primer pairs. Data were analyzed with the ΔCt method and gene expression was normalized to average expression of Gapdh and Hmbg1. Primers for the genes of interest were obtained from Bio-Rad Laboratories (Table 1). Collagen type 1 alpha 1 qMuCED0044222 Col2a1 Collagen type 2 alpa 1 qMuCID0006546

Histology
Right hind limbs were fixed in PFA for 2 days, stored in PBS at 4  C overnight, and then paraffin embedded. Sections were cut to 4 µm, collected at 100 µm intervals, and stained with Safranin O (Saf-O)−Fast Green as described previously [5] to evaluate proteoglycan content and pathological changes such as cartilage degradation.

Histomorphometry
Histomorphometric analyses were performed using NIS-Elements software (Nikon, AR 5.11.03). The Saf-O+ area was measured impartially by the NIS software using identical thresholding parameters between all images (pixels falling below the threshold intensity of 20 were uncounted) by detecting the total red (Saf-O+) pixel area. The total cartilage area was manually selected with border detection assistance using the NIS software's thresholding tools. Saf-O+ chondrocytes were manually counted for each femoral condyle and tibial plateau and graphed as average Saf-O+ chondrocytes and number of Saf-O+ chondrocytes per mm 2 of total AC. Chondrocytes were considered Saf-O+ if they were surrounded by red-stained matrix.

Gait analysis
All measurements were performed at Northwestern University's Behavioral Phenotyping Core one-month post-transplantation with an Institutional Animal Care and Use Committee approved animal protocol. Walking speed and gait analysis were assessed using a DigiGait Imaging System (Mouse Specifics Inc.). Briefly, the mice were tested separately within the motorized treadmill chamber at speeds of 10 cm/s, 17 cm/s, and 24 cm/s. Completion of each speed was accomplished when the mouse was able to run uninterrupted for a minimum of 4 s (the amount of time required for DigiGait software to capture sufficient data for analysis) on two separate trials. Each mouse was provided a minimum of five attempts to complete each speed. The percent of successful trials at each speed was recorded as running ability. The stride length, swing duration, and paw area gait parameters were obtained from the data generated using the DigiGait software. Aged mice that did not complete a single trial at a given speed, i.e., those with the greatest functional defects, were therefore unable to generate gait data for analysis. These gait parameters, relevant to aging-related diseases, were chosen in advance to eliminate selection bias from the 20+ gait parameters generated by the DigiGait software.

Statistics
Statistical analyses were carried out using the SigmaPlot (Jandel Scientific, v14.0) software package. The twotailed unpaired Welch's unequal variance t-test, twotailed Student's t-test, or the Mann-Whitney rank sum test were used where appropriate for direct comparison(s) between treatment and control groups. A one-tailed Student's t-test was used to detect functional improvements following MDSPC transplantation. All values are expressed as the mean ± SEM., and p < 0.05 was considered statistically significant

Articular cartilage of naturally aged mice shows agingrelated imbalances associated with local catabolic and anabolic activity
To identify changes in the AC of naturally aged (NA) mice, we compared gene expression in PBS-injected NA control (NA-C; 24 months old) to young mice (5-6 months old). Expression of pro-inflammatory cytokine and senescence-associated secretory phenotype (SASP) genes, Il1a and Tnf-which promote age-related pathologies associated with OA and enhance extracellular matrix (ECM) proteinases [6]-was significantly upregulated in NA-C mice compared to young mice (Fig.  1A). NA-C mice also exhibited a prominent increase in expression of catabolic factors that drive ECM degradation and cleave collagen type II and aggrecans (Fig. 1B), specifically, Mmp3, Mmp13, and Adamts5 [7]. The expression of genes responsible for cartilage tensile strength and tissue repair (Col2a1) and cartilage growth (Bgn) [8] were also significantly decreased in NA-C mice compared to young (Fig. 1C).

Systemic transplantation of young MDSPCs ameliorates articular cartilage homeostasis in naturally aged mice
We next examined whether systemic transplantation of young MDSPCs-isolated from 3-week-old miceameliorates these OA-like changes. In NA mice intraperitoneally injected with MDSPCs (NA-IP) (Fig.  1D), Il1a and Tnf, were significantly downregulated in the AC compared to NA-C mice (Fig. 1E). Expression of Mmp3 and Mmp13 were also significantly decreased, with Adamts5 exhibiting a strong, though not statistically significant (p = 0.065), decrease (Fig. 1F). Moreover, compared to NA-C mice, NA-IP mice showed significantly increased expression of genes encoding factors involved in cartilage tensile strength and tissue repair (Col2a1), resilience (Acan), chondrocyte proliferation and differentiation (Vcan), and growth (Bgn) [8] (Fig. 1G). Similar to previous reports, we did not observe a difference between groups in mRNA expression of Col1a1 [9]. Aging chondrocytes exhibit senescent phenotypes, including telomere shortening [10]. Notably, expression of the telomere protection and pro-longevity gene [11], Pot1b, was significantly increased in NA-IP mice. However, expression levels of other stress response and DNA damage genes-including Gpx4, Sod1, or Gadd45a-remained unchanged (Fig. 1H-I). .

Systemic transplantation of young MDSPCs rejuvenates articular cartilage histology and improves mobility in aged mice
To determine the effects of MDSPC treatment on AC histopathology, we stained the knee joints with Saf-O to detect ECM proteoglycans, which are progressively lost during aging. The femoral condyle ( Fig. 2A) and tibial plateau (Fig. 2B) of the AC revealed that while total cartilage area was similar (Fig. 2C), 51% of cartilage in NA-IP mice was Saf-O+ compared to just 21% in NA-C mice (Fig. 2D). NA-IP mice also had greater Saf-O+ chondrocyte numbers (Fig. 2E) and density (Fig. 2F). To identify the functional impact from molecular-and tissue-level rejuvenation, gait parameters were measured one month after MDSPC transplantation using DigiGait. MDSPC treatment significantly increased stride length (Fig. 2G), swing duration (Fig. 2H), and paw area (Fig.  2I) compared to controls. These metrics decline in animal models of age-related movement disorders including arthritis, chronic joint pain, Parkinson's disease, and diabetic peripheral neuropathy [12][13][14][15]. NA-IP mice were also significantly more capable of running at higher speeds (Fig. 2J) than NA-C mice.

DISCUSSION
Together, our findings demonstrate, for the first time, the rejuvenating effects of systemic transplantation of young MDSPCs on aged AC. This treatment reduced proinflammatory factors and ECM proteinases while increasing anabolic factors and proteoglycan content, thereby reversing age-associated degradation. Thus, overcoming these local negative regulators resulted in AC regeneration and significant functional improvements in gait metrics and speed. The mechanism(s) underlying rejuvenation remain speculative and are the main focus of our ongoing studies. However, these results reinforce the paracrine mechanism suggested by our previous work in which systemic transplantation of young MDSPCs significantly extended the lifespan and healthspan of progeroid mice [3]. We hypothesize that MDSPCs orchestrate their pro-regenerative effects by inhibiting AC degeneration and regulating chondrocyte viability and responsiveness to extrinsic factors, which may restore the AC catabolic/anabolic equilibrium in aged hosts to a more "youthful" state. Other possible mechanisms include telomere protection and modulation of the inflammatory SASP cascade. A limitation of this study includes controls not being transplanted with a biologic material, as done in our previous study [3] which demonstrated the necessity of young MDSPCs. It is also possible that due to the systemic nature of this cell therapy, the functional improvements are multifactorial, involving AC, muscle, nerve, and/or bone. Furthermore, translation from murine models to human OA therapies can be difficult and additional safety studies for cell transplantation will need to be performed. However, application of MDSPCs and/or their secreted factors may represent an attractive intervention for aged and OA patients to rejuvenate cartilage and increase mobility.