Camilla Brantsing

Proliferation and differentiation potential of chondrocytes from osteoarthritic patients.

Autologous chondrocyte transplantation (ACT) has been shown, in long-term follow-up studies, to be a promising treatment for the repair of isolated cartilage lesions. The method is based on an implantation of in vitro expanded chondrocytes originating from a small cartilage biopsy harvested from a non-weight-bearing area within the joint. In patients with osteoarthritis (OA), there is a need for the resurfacing of large areas, which could potentially be made by using a scaffold in combination with culture-expanded cells. As a first step towards a cell-based therapy for OA, we therefore investigated the expansion and redifferentiation potential in vitro of chondrocytes isolated from patients undergoing total knee replacement. The results demonstrate that OA chondrocytes have a good proliferation potential and are able to redifferentiate in a three-dimensional pellet model. During the redifferentiation, the OA cells expressed increasing amounts of DNA and proteoglycans, and at day 14 the cells from all donors contained type II collagen-rich matrix. The accumulation of proteoglycans was in comparable amounts to those from ACT donors, whereas total collagen was significantly lower in all of the redifferentiated OA chondrocytes. When the OA chondrocytes were loaded into a scaffold based on hyaluronic acid, they bound to the scaffold and produced cartilage-specific matrix proteins. Thus, autologous chondrocytes are a potential source for the biological treatment of OA patients but the limited collagen synthesis of the OA chondrocytes needs to be further explained.

Notch1, Jagged1, and HES5 are abundantly expressed in osteoarthritis.

Background: Notch signalling controls differentiation and proliferation in various cell types and is associated with several diseases. We investigated the localization and regulation of several Notch markers in human osteoarthritic (OA) cartilage as well as identified genes controlled by Notch signalling. Methods: Immunolocalization and real-time PCR analysis of Notch markers in healthy and OA articular cartilage were performed. Genes regulated by Notch signalling were studied using microarray. Cytokine-induced transcription of Notch markers was analyzed using real-time PCR and its effect on cellular localization of the intracellular domain of Notch1 (NICD1) was investigated using immunohistochemistry, subcellular fractionation, and transfection. The effect of NFĸB activation on HES5 transcription was studied using the NFĸB inhibitor pyrrolidine dithiocarbamate. Results: Notch signalling was activated in OA cartilage and Notch1, Jagged1, and HES5 were abundantly expressed compared to healthy cartilage. Notch signalling regulated the expression of several genes associated with OA, like interleukin-8, lubricin, CD10, matrix metalloproteinase-9, and bone morphogenetic protein-2. Cytokines significantly affected the expression of several Notch markers and repressed expression of HES5, but did not affect the cellular localization of NICD1. Conclusion: Notch signalling is dysregulated in OA, inducing and repressing transcription of genes that could potentially partly contribute to the OA phenotype.

Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink

Cartilage lesions can progress into secondary osteoarthritis and cause severe clinical problems in numerous patients. As a prospective treatment of such lesions, human-derived induced pluripotent stem cells (iPSCs) were shown to be 3D bioprinted into cartilage mimics using a nanofibrillated cellulose (NFC) composite bioink when co-printed with irradiated human chondrocytes. Two bioinks were investigated: NFC with alginate (NFC/A) or hyaluronic acid (NFC/HA). Low proliferation and phenotypic changes away from pluripotency were seen in the case of NFC/HA. However, in the case of the 3D-bioprinted NFC/A (60/40, dry weight % ratio) constructs, pluripotency was initially maintained, and after five weeks, hyaline-like cartilaginous tissue with collagen type II expression and lacking tumorigenic Oct4 expression was observed in 3D -bioprinted NFC/A (60/40, dry weight % relation) constructs. Moreover, a marked increase in cell number within the cartilaginous tissue was detected by 2-photon fluorescence microscopy, indicating the importance of high cell densities in the pursuit of achieving good survival after printing. We conclude that NFC/A bioink is suitable for bioprinting iPSCs to support cartilage production in co-cultures with irradiated chondrocytes.

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.

Cardiac Insulin-like Growth Factor I and Growth Hormone Receptor Expression in Renal Hypertension

The aim of the present study was to investigate the role of insulin-like growth factor I in the development of cardiac hypertrophy in two-kidney, one clip hypertension by relating growth hormone receptor and insulin-like growth factor I receptor mRNA levels to insulin-like growth factor I gene transcription using a solution hybridization/RNase protection assay. Two-kidney, one clip hypertension was induced in male Wistar rats, and experiments were performed 2, 4, 7, and 12 days after surgery. Systolic blood pressure was elevated 2, 7, and 12 days after clipping (P < .001). Left ventricular weights were increased 2, 4, 7, and 12 days after surgery (P < .01). Associated with the rise in blood pressure, left ventricular insulin-like growth factor I mRNA was increased 2, 7, and 12 days after surgery (P < .01). Furthermore, growth hormone receptor and insulin-like growth factor I receptor gene expression increased specifically in the left ventricle of renal hypertensive rats (P < .05 and P < .001, respectively). Left ventricular growth hormone receptor mRNA peaked 7 days after induction of renal artery stenosis. These results show that insulin-like growth factor I, growth hormone receptor, and insulin-like growth factor I receptor mRNA increase in the pressure-overloaded left ventricle of two-kidney, one clip rats, suggesting a role for insulin-like growth factor I and the growth hormone/insulin-like growth factor I axis in the development of cardiac hypertrophy.

Human serum for culture of articular chondrocytes.

In the field of cell and tissue engineering, culture expansion of human cells in monolayer plays an important part. Traditionally, cell cultures have been supplemented with serum to support attachment and proliferation, but serum is a potential source of foreign protein contamination and viral protein transmission. In this study, we evaluated the use of human serum for experimental human articular chondrocyte expansion and to develop a method for preparation of large volumes of high-quality human serum from healthy blood donors. Human autologous serum contained high levels of epidermal-derived growth factor and platelet-derived growth factor-AB and supported proliferation up to 7 times higher than FCS in primary chondrocyte cultures. By letting the coagulation take place in a commercially available transfusion bag overnight, up to 250 ml of growth factor-rich human serum could be obtained from one donor. The allogenic human serum supported high proliferation rate without loosing expression of cartilage-specific genes. The expanded chondrocytes were able to redifferentiate and form cartilage matrix in comparable amounts to autologous serums. In conclusion, the transfusion bags allow preparation of large volumes of growth factor-rich human serum with the capacity to support in vitro cell expansion. The data further indicate that by controlling the coagulation process there are possibilities of optimizing the release of growth factors for other emerging cell therapies.

Involvement of INK4A gene products in the pathogenesis and development of human osteosarcoma

The INK4A tumor suppressor gene plays a crucial role in the regulation of the G1 cell cycle phase. It encodes two transcripts, p16 and p14 alternate reading frame (ARF), involved in retinoblastoma protein (pRb)‐ and p53‐ cell growth control pathways, respectively.

Notch and HES5 are regulated during human cartilage differentiation

The molecular mechanisms of cartilage differentiation are poorly understood. In a variety of tissues other than cartilage, members of the basic helix-loop-helix (bHLH) family of transcription factors have been demonstrated to play critical roles in differentiation. We have characterized the human bHLH gene HES5 and investigated its role during chondrogenesis. Blockage of the Notch signaling pathway with a γ-secretase inhibitor has demonstrated that the human HES5 gene is a downstream marker of Notch signaling in articular chondrocytes. Markers for the Notch signaling pathway significantly decrease during cartilage differentiation in vitro. Cell proliferation assayed by using BrdU has revealed that blockage of Notch signaling is associated with significantly decreased proliferation. Northern blot and reverse transcription/polymerase chain reaction of a panel of various tissues have shown that HES5 is transcribed as a 5.4-kb mRNA that is ubiquitously expressed in diverse fetal and adult tissues. Articular cartilage from HES5−/− and wild-type mice has been analyzed by using various histological stains. No differences have been detected between the wild-type and HES5−/− mice. Our data thus indicate that the human HES5 gene is coupled to the Notch receptor family, that expression of Notch markers (including HES5) decreases during cartilage differentiation, and that the blockage of Notch signaling is associated with significantly decreased cell proliferation.

Footprint-Free Human Induced Pluripotent Stem Cells From Articular Cartilage With Redifferentiation Capacity: A First Step Toward a Clinical-Grade Cell Source

Human induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine; however, clinical applications of iPSCs are restricted because of undesired genomic modifications associated with most reprogramming protocols. We show, for the first time, that chondrocytes from autologous chondrocyte implantation (ACI) donors can be efficiently reprogrammed into iPSCs using a nonintegrating method based on mRNA delivery, resulting in footprint‐free iPSCs (no genome‐sequence modifications), devoid of viral factors or remaining reprogramming molecules. The search for universal allogeneic cell sources for the ACI regenerative treatment has been difficult because making chondrocytes with high matrix‐forming capacity from pluripotent human embryonic stem cells has proven challenging and human mesenchymal stem cells have a predisposition to form hypertrophic cartilage and bone. We show that chondrocyte‐derived iPSCs can be redifferentiated in vitro into cartilage matrix‐producing cells better than fibroblast‐derived iPSCs and on par with the donor chondrocytes, suggesting the existence of a differentiation bias toward the somatic cell origin and making chondrocyte‐derived iPSCs a promising candidate universal cell source for ACI. Whole‐genome single nucleotide polymorphism array and karyotyping were used to verify the genomic integrity and stability of the established iPSC lines. Our results suggest that RNA‐based technology eliminates the risk of genomic integrations or aberrations, an important step toward a clinical‐grade cell source for regenerative medicine such as treatment of cartilage defects and osteoarthritis.

Induction of Growth Hormone Receptor and Insulin-Like Growth Factor-I mRNA in Aorta and Caval Vein During Hemodynamic Challenge

Induction of two-kidney, one clip hypertension (renal hypertension) is characterized by a slow increase in left ventricular tension and aortic wall stress, as opposed to aortocaval fistula or shunt volume overload, which induces a marked and rapid onset of wall stress in the caval vein and right ventricle. In the present study, we applied hemodynamic challenge to study the growth response involving gene expression of insulin-like growth factor-I (IGF-I) and growth hormone receptor (GH-R) mRNA in aorta and caval vein. Volume overload and pressure overload were induced in Wistar rats by means of shunt and renal hypertension, respectively. Systolic pressure was measured before excision of the great vessels, which was performed between 2 and 12 days postoperatively. Aortic and caval vein IGF-I and GH-R mRNA expressions were measured by means of a solution hybridization assay, and the caval vein was analyzed for IGF-I protein by immunohistochemistry. In the volume-distended but not pressurized caval vein in shunt rats, verified by telemetry recordings, there was an eightfold increase in IGF-I and 3.5-fold increase in GH-R mRNA at day 4 versus control. The IGF-I protein appeared to be localized in smooth muscle cells. In the aorta of the renal hypertension group, changes were of a slower onset. At day 7, there was a fourfold increase in IGF-I and five-fold increase of GH-R mRNA expressions versus sham-operated rats. Both the shunt caval vein and renal hypertension aorta showed evidence of a structural adaptation of the growth response. The present study suggests that acute elevation in vascular wall stress is an important triggering factor for overexpression of IGF-I and GH-R mRNA in great vessels. The growth hormone/insulin-like growth factor axis may be an important link in mediating structurally adaptive growth responses in the blood vessel wall.