Traceability of injected MSCs in IVD patients after 8 months

Low back pain has high impact on Western world societies, both physically through the
morbidity of afflicted individuals and financially through loss of productivity and increasing
health care cost. Intervertebral disc (IVD) degeneration is considered to be one of the
major causes of low back pain and is characterized by dysfunctional cells that cause the
loss of proteoglycan production.
Currently the first line of treatment for painful degenerative disc disease is non‐surgical,
including physiotherapy and analgesics. Surgical treatment is considered in refractory cases
and options include spinal fusion or IVD prostheses.
At recent years, cell therapy based methods have been discussed as alternative/
complementary treatment options. The goal would be to replace or enhance the function
of certain cell types with the goal to influence the function of the IVD. Injection of MSCs
into IVDs in different animal models has been performed with positive outcome
Transplantation of MSC´s to the IVD has also been performed in a few limited patient
cohort studies, however the fate of the cells has not been investigated. Currently,
both local and systemic administration applications are used to transplant human MSCs or
hemapoetic stem cells into different organs and for different pathologies. For
the evaluation of novel cell therapy applications it would be beneficial to be able to trace
the injected cells for localization purposes but also for evaluation of function. In the adult
human, the total‐body iron content ranges between 2‐4 g. One third of the iron deposits in
the human body are stored in the liver, spleen and bone marrow, two‐third are bound to
circulating hemoglobin molecules and about 0.1 percent of the total iron pool is present in
the peripheral blood circulation (plasma) where it is bound to transferrin. The up‐
take of the Fe3+‐transferrin complex of cells is carried out by active transport over the cell
membrane. Other systems for iron –uptake of cells include endocytosis and
phagocytosis.

Super‐paramagnetic iron nanoparticles (SPIOs, i.e. Endorem®) which are non‐toxic, have
earlier been used clinically for the detection of tumors in e.g. the lymphatic system using
magnetic resonance imaging (MRI). SPIOs have been used as cell tracers in previous in
vitro studies and in vivo studies (animal models and human). However, since SPIOs approved for clinical use in humans are no longer commercially available other iron compounds are of interest as options to be used as cell tracers. The iron sucrose compound Venofer® is a pharmaceutical, clinically approved drug, commonly administered to patients with e.g. with iron deficiency conditions. The iron in this
molecular complex is organized in a way that resembles the natural physiologically
occurring compound ferritin (intracellular form of iron in humans) and has
desirable properties for a cell tracer. Iron sucrose has been shown to function as an intra
cellular compound for cell tracing purposes both in vitro and in vivo. In one of
these studies the effect on cell viability and chondrogenic differentiation of human MSCs
labelled with iron sucrose was investigated in vitro. It was observed that iron sucrose had
no major negative cell effects but somewhat affected the onset of differentiation into the
chondrogenic lineage. Further, in an in vivo study (lapine model), after injection it was
demonstrated that human MSCs labelled with iron sucrose were viable and could be
traced up to three months post‐injection.
In the present study, autologous human MSCs were labelled prior to injection into
degenerated IVDs in patients with disc degeneration and chronic low back pain.
Biomarkers used in the present study for mapping of cellular processes in the injected IVD
tissue regions include proliferating cell nuclear antigen (PCNA) a biomarker expressed
during DNA replication during cell proliferation and the sex determining region Y box 9
(SOX9), a transcription factor and an important key regulator in chondrogenesis. SOX9 is
expressed during the chondrogenesis e.g. during normal limb formation and interacts with
bone morphogenic proteins signaling (BMPs) . Collagen 2A1 (COLL2A1) is a major
component of the extra cellular matrix (ECM) in cartilage, expressed when collagen is
produced and deposited by e.g. chondrocytes or chondrocyte like cells and was also
investigated. CD68 is a marker for a transmembrane glycoprotein highly expressed in
human macrophages and monocytes .

The aim of the present study was to investigate the traceability, potential proliferation and
differentiation of autologous, bone marrow derived, iron labelled MSCs that were injected
into IVDs, in patients with disc degeneration and chronic low back pain.

Detection of iron labelled cells in explanted IVD tissues

Iron labelled cells (blue color) were detected in explanted degenerated IVD tissue samples
in several tissue preparations for the 3 IVDs injected 6‐12 months prior explanting. The
cells were detected in large cell clusters and/or as solitary cells. A higher cell density was
observed in the areas with cell clusters with iron labeled cells compared to in areas of
native degenerated IVD tissues. Further, extra cellular iron particles were observed in
some areas in IVD tissues from the donors 1‐3 and in small areas in tissue samples from
donor 4 (2.5 years post injection).

FIGURE 1
A few regions with clusters of iron labelled cells were identified in the degenerated IVD
tissue sections/samples from donor 1‐2 and were referred to as regions of interest (ROIs).
These IVD samples were analyzed in more detail by histology and IHC using consecutive
serially numbered sections. All the presented histology and IHC images were taken in the
same corresponding regions to ROIs.

Extra cellular matrix accumulation
Accumulation of ECM was observed in the investigated ROI areas and the presence of
glucosaminoglycans was seen in close proximity to the corresponding area were the iron
labeled cells were detected. Expression of Coll2A1 was detected in close proximity to
solitary cells in the ROI areas.

FIGURE 2‐3
Cell proliferation and differentiation
A low number of PCNA and SOX9 positive cells were detected in the ROI areas in the
investigated samples (2/2 donors).

FIGURE 3
Cell viability
By TUNEL assay apoptotic cells were detected in ROIs in a relatively low number in one of
the investigated samples (donor 1) but could not be detected in donor 2.

FIGURE 4
CD68 expression
A low number of cells positive for CD68 were detected in the ROI area for donor 2 but not
in donor 1.

FIGURE 5
Presence of calcium deposits/bone formation
Small calcium deposits (black dots) were observed by von Kossa staining in ROI in donor 1
and in non ROI areas of the IVD samples indicating possible early bone formation.

DISCUSSION
This is to our knowledge the first study investigating the fate of injected autologous MSCs
in explanted IVD tissue samples from patients where MSCs have been injected in an
attempt to halt the progress of disc degeneration and influence chronic low back pain.
Injected iron labeled cells (pre‐labeled before the injection) were detected by Preussian
blue staining in cell clusters and/or as solitary cells in identified IVD tissue samples of
patients injected 6‐12 months prior explantation. In the patient injected 2.5 years prior to
the disc explantation only extracellular iron particles could be detected. In the IVD tissue
regions with iron‐labelled cells (ROIs) high cellular activity was detected by the cell
proliferation marker PCNA and the chondrogenic lineage marker SOX9. Further, ECM
accumulation was observed by COLL2A1 expression in the investigated ROIs. This indicates
viable cells and an on‐going cellular activity in regions with injected cells and/or their
progeny present. It is further likely that the injected cells signal and influence the resident
disc cell populations and /or local progenitor cell populations of the degenerated IVD,
which may contribute to the observed cellular activity in the investigated regions. This
assumption is made based on a number of in vitro studies demonstrating positive
interaction between disc cells and MSCs where e.g. positive effects on ECM accumulation
has been observed in co‐culture of MSCs and IVD cells.
Further, the injected MSCs might also have influenced local progenitor cell populations
that have been reported to be present in non‐degenerated IVDs (animals) and in
degenerated human IVDs .
The findings of a small number of apoptotic cells and remaining extracellular iron‐staining
in some tissue preparations in the present study indicate that a certain percentage of the
injected cells will not survive over time. This might be due to several reasons e.g. the
injection procedure per se, insufficient nutrition and/or poor acclimatizing to the harsh
microenvironment of the degenerated disc e.g. low pH. The extracellular iron deposits may also simply be caused by leakage of intracellular iron to the environment
over time.
A very low amount of calcium deposits were detected in one of the investigated samples.
However, this was also observed in the non‐ROI areas of this patient. Only a low number
of CD68 positive cells were observed in one of the investigated samples (donor 2), which
indicate that no strong inflammatory response were triggered by the MSCs.
The main limitation of this study is the limited amount of tissue samples collected in small
pieces from few subjects, not allowing either overall distribution or quantitative analysis
within the IVD´s to be performed. Further, it was not possible in this study to determine if
the cellular activity observed in the ROIs was derived from the injected MSCs per se
(and/or their progeny) or if the local resident disc cells and/or progenitor cell populations
in the IVD were activated by paracrine signaling deriving from the MSCs.
This study demonstrates that MSCs, labelled with iron sucrose, injected into degenerated
IVDs can be traced at least 6‐12 months in explanted IVD tissue samples. The detected
cellular activity and morphology of the traceable cells indicate that the injected cells and
/or their progeny have survived since the cells were both found in large clusters and as
solitary cells which were distributed to different parts of the IVD (as observed in the
histology samples) at varying distances from the clusters. In cartilaginous tissues, the iron
levels are very low compared to the main iron deposits which in the in the human body are
stored in the e.g. liver, spleen and bone marrow. Therefore the presence of large clusters
with strongly iron positive cells in the IVD tissues can be considered to be the injected iron
labeled cells and/or their progeny.
The distribution of the cells could be due to cellular migration or by the injection per se.
Further, the results indicated that the injected MSCs had differentiated into the
chondrogenic lineage and/or stimulated native cells to produce extra cellular matrix
proteins.
The results from this study contribute to the understanding of the fate of MSCs after
injection into IVDs in patients and the findings may be of further interest for the
development of stem cell therapy for other degenerated tissues.

REFERENCE:

  1. The traceability of mesenchymal stromal cells after injection into degenerated discs in patients with low back pain. Helena Barreto Henriksson, Nikolaos Papadimitriou, Daphne Hingert, Adad Baranto, Anders Lindahl, Helena Brisby DOI: 10.1089/scd.2019.0074
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