Abstract

Background

   Mesenchymal stromal cells (MSCs) have been studied with increasing
   intensity as clinicians and researchers strive to understand the
   ability of MSCs to modulate disease progression and promote tissue
   regeneration. As MSCs are used for diverse applications, it is
   important to appreciate how specific physiological environments may
   stimulate changes that alter the phenotype of the cells. One need for
   neuroregenerative applications is to characterize the spectrum of MSC
   responses to the cerebrospinal fluid (CSF) environment after their
   injection into the intrathecal space. Mechanistic understanding of
   cellular biology in response to the CSF environment may predict the
   ability of MSCs to promote injury repair or provide neuroprotection in
   neurodegenerative diseases.

Methods

   In this study, we characterized changes in morphology, metabolism, and
   gene expression occurring in human adipose-derived MSCs cultured in
   human (hCSF) or artificial CSF (aCSF) as well as examined relevant
   protein levels in the CSF of subjects treated with MSCs for amyotrophic
   lateral sclerosis (ALS).

Results

   Our results demonstrated that, under intrathecal-like conditions, MSCs
   retained their morphology, though they became quiescent. Large-scale
   transcriptomic analysis of MSCs revealed a distinct gene expression
   profile for cells cultured in aCSF. The aCSF culture environment
   induced expression of genes related to angiogenesis and
   immunomodulation. In addition, MSCs in aCSF expressed genes encoding
   nutritional growth factors to expression levels at or above those of
   control cells. Furthermore, we observed a dose-dependent increase in
   growth factors and immunomodulatory cytokines in CSF from subjects with
   ALS treated intrathecally with autologous MSCs.

Conclusions

   Overall, our results suggest that MSCs injected into the intrathecal
   space in ongoing clinical trials remain viable and may provide a
   therapeutic benefit to patients.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s13287-021-02241-9.

   Keywords: Mesenchymal stromal cell, Gene expression, Neuroinflammation,
   Growth factors, Cerebrospinal fluid, Amyotrophic lateral sclerosis,
   Human studies

Introduction

   Mesenchymal stromal cells (MSCs) are increasingly being employed in
   clinical trials to treat neurodegenerative diseases despite a lack of
   mechanistic investigations into the phenotypes of MSCs when injected
   into the central nervous system (CNS) compartment. For many current
   trials for neurological diseases, MSCs are extracted from adipose
   tissues or bone marrow, expanded in vitro, and then injected into the
   intrathecal cerebrospinal fluid (CSF) of the CNS to bypass the
   blood-brain barrier. The hypothesis, based on pre-clinical in vitro and
   animal models, is that MSCs will secrete factors that have a
   therapeutic effect on neurons and the surrounding microenvironment.
   MSC-based therapy is attractive because most neurodegenerative diseases
   involve multi-faceted pathologies, and MSCs have been shown to
   favorably modulate the majority of these pathological processes.
   However, pre-clinical models often recapitulate neither the intrathecal
   microenvironment nor the preparation of MSCs that would be administered
   to a clinical trial patient, hampering responsible translation of MSCs
   into clinical use.

   Intrathecal CSF represents a uniquely challenging environment for
   cultured MSCs: it has a lower partial pressure of oxygen than media in
   culture conditions (~ 70–80 mmHg in CSF) [[47]1], undergoes age- and
   disease-related changes in protein composition [[48]2–[49]4], and
   contains less protein compared to plasma, with CSF containing primarily
   albumin and brain-derived factors [[50]5]. When MSCs are cultured in
   vitro, cells are grown at normoxia and the growth media must be
   supplemented with nutrient-rich human platelet lysate (hPL) to support
   healthy proliferation prior to their harvest and implantation. When
   cultured without serum or hPL, or in hypoxic conditions, MSCs have been
   shown to rapidly undergo apoptosis and risk chromosomal instability
   [[51]6–[52]8]. However, previous studies—albeit utilizing diluted CSF
   formulations—have shown that MSCs can remain viable and increase the
   presence of nutritional factors within CSF [[53]9–[54]11]. So how are
   researchers and clinicians to harmonize this data to balance the risks
   with the purported benefits of MSC therapy?

   In addition to the above-mentioned weaknesses of pre-clinical models,
   they often also fail to account for the preparation of MSCs for
   clinical use, which has the potential to drastically impact the
   phenotype of MSCs. The field of cell therapy is becoming increasingly
   cognizant of how culture conditions affect MSCs. Understandably, most
   preclinical research to date has been conducted using research-grade
   reagents, such as media supplemented with fetal bovine serum, that are
   not permitted for cells destined for human use. This distinction is
   important to note as numerous studies demonstrated that MSCs grown in
   compliance with current good manufacturing practices (GMP) versus
   non-GMP conditions (and even within varying GMP-compliant conditions)
   display disparate phenotypes characterized by different proliferation
   rates, differentiation potentials, and immunomodulatory potentials
   [[55]12–[56]15]. At present, it is unclear how in vitro manufacturing
   conditions ultimately influence the response of MSCs to the CSF
   environment. Furthermore, how the CSF microenvironment, with its lower
   oxygen and nutrient levels, impacts MSCs cultured for clinical use is
   also unknown.

   To address these gaps in understanding, and to guide stakeholders in
   MSC-based treatment of neurodegenerative diseases, we aimed to
   characterize the morphological, metabolic, and transcriptomic changes
   observed when MSCs are incubated in undiluted human (hCSF) or
   artificial CSF (aCSF) as compared to control media (CM; containing
   GMP-grade hPL) and platelet lysate-free (PLF) media. We hypothesized
   that the phenotype of MSCs cultured in undiluted CSF formulations would
   mimic that of MSCs cultured in conditions devoid of platelet lysate,
   which are known to be stressful for MSCs. To extend prior work and to
   most accurately reflect the clinical application, our studies
   emphasized the use of clinical-grade MSCs cultured in GMP-grade
   reagents to recapitulate the phenotype of cells that are currently used
   to treat patients [[57]16–[58]18].

   To supplement our in vitro results, we undertook a secondary set of
   experiments in which we analyzed CSF collected from subjects in a phase
   I clinical trial at the Mayo Clinic investigating the use of
   intrathecal MSCs to treat amyotrophic lateral sclerosis (ALS) [[59]17],
   a neurodegenerative disorder that leads to a progressive and fatal
   paralysis. These secondary experiments were designed to examine the
   protein levels of therapeutically-relevant growth and immunomodulatory
   factors within the CSF of patients treated with MSCs for additional
   evidence to support the potential therapeutic mechanism of action of
   MSCs within the intrathecal space.

Materials and methods

Mesenchymal stromal cells and cerebrospinal fluid

   With approval from the Mayo Clinic Institutional Review Board (IRB),
   MSCs were obtained from four consenting, healthy patients who underwent
   elective removal of subcutaneous adipose tissue either by
   lipoaspiration [male/41 years (M1), female/32 years (F1), male/54 years
   (M2)] or by surgical biopsy [a female (F2) bariatric patient who
   donated cells as medical waste so age was not collected]. Samples were
   processed to isolate and expand MSCs as previously described [[60]7,
   [61]19, [62]20]. Cells were stored in liquid nitrogen until use. All
   patient information was kept confidential and all identifiers removed
   prior to any studies.

   hCSF that otherwise would be disposed as medical waste was obtained
   from consenting patients treated with lumbar puncture in the
   Hydrocephalus Clinic at the Mayo Clinic with approval from the Mayo
   Clinic IRB.

Solutions

   CM used for studies consisted of Advanced Minimal Essential Medium
   (MEM) standard culture medium with 5% PLTmax (platelet lysate solution,
   Mill Creek Life Sciences), 2 U/mL heparin (hospital pharmacy),
   2 mM L-glutamine (Invitrogen), and antibiotics (100 U/ml penicillin,
   100 g/ml streptomycin). The PLF media was made using Advanced MEM with
   2 mM L-glutamine and antibiotics. The aCSF formulation comprised the
   following: distilled water with NaCl (124 mM), KCl (2.5 mM), NaHCO[3]
   (26 mM), NaH[2]PO[4]-H[2]O (1.25 mM), MgSO[4] (1 mM), CaCl[2] (2 mM),
   D-glucose (60 mg/dL), and human albumin (30 mg/dL). The aCSF had a pH
   of 7.4 and was warmed along with the other solutions before use.

Immunocytochemistry on MSCs cultured in human CSF

   MSCs were seeded in CM at 3000 cells/cm^2 in 6-well plates for 24 h on
   glass coverslips prior to introduction of fresh CM, PLF media, or hCSF
   for 24–48 h. Upon removal of the media, cells were stained with
   pre-warmed 100 nM MitoTracker CMX-Ros solution (Invitrogen, Carlsbad,
   CA) for 20 min at 37 °C. Cells were then fixed for 10 min in a
   pre-warmed solution of 4% paraformaldehyde in Dulbecco’s phosphate
   buffer solution (D-PBS). Following fixation, cells were washed 3x for
   5 min in 1x D-PBS, permeabilized for 10 min with phosphate buffered
   solution with Triton X-100 (PBS-T—0.1% Triton X-100 in 1x PBS), and
   washed again 3x for 5 min in D-PBS. Cells were blocked for 30 min with
   1% bovine serum albumin in 1x D-PBS. Cells were incubated for 60 min
   with Alexa Fluor 488 phalloidin (1:100, Invitrogen, Carlsbad, CA).
   After washing in 1x D-PBS and removal of excess liquid, the cover slips
   were mounted onto slides using ProLong Gold antifade reagent with DAPI
   (Invitrogen, Carlsbad, CA) and left to cure overnight. Slides were
   imaged with an AxioScope fluorescent microscope at × 40 magnification.

Metabolic activity of MSCs in aCSF

   MSCs were seeded in CM at 2500 cells/cm^2 in 12-well plates and grown
   to about 80% confluency prior to introduction of fresh CM, PLF media,
   or aCSF for 24–96 h. At each time point, culture solutions were
   collected and cell viability analyzed using an MTS assay following the
   manufacturer’s instructions (Promega, Madison, WI). Absorbance of each
   sample was read at 490 nm using a plate reader and corrected using
   absorbance values of empty control wells. Metabolic activity for PLF
   media- and aCSF-treated cells was assessed in comparison to the
   absorbance of wells containing control CM-treated cells.

Transcriptomic analyses

   MSCs were seeded with about 3000 cells/cm^2 in 6-well plates and
   monitored until approximately 80% confluent. Cells were incubated in
   with fresh CM, PLF media, or aCSF for 24 or 48 h before harvesting.
   Cells from each line were plated in three replicate wells per condition
   per time point to yield 18 wells total per line—6 treated with CM, 6
   treated with PLF media, 6 treated with aCSF—with half the wells treated
   for 24 h and half treated for 48 h. Total RNA was isolated per the
   manufacturer’s instructions using the miRNeasy Mini Plus Kit (Qiagen,
   Germantown, MD). RNA libraries were prepared according to the
   manufacturer’s instructions for the TruSeq RNA Sample Prep Kit v2
   (Illumina, San Diego, CA) that uses oligo dT magnetic beads to enrich
   poly-A mRNA. Polymerase chain reaction (PCR) was used to enrich the
   resulting cDNA fragments using Illumina TruSeq PCR primers. The
   concentration and size distribution of the completed library was
   determined using a Fragment Analyzer (AATI, Ankeny, IA) and Qubit
   fluorometry (Invitrogen, Carlsbad, CA). The cDNA library was sequenced
   at 30–50 million fragment reads per sample following Illumina’s
   standard protocol using the Illumina cBot and HiSeq 3000/4000 PE
   Cluster Kit. The flow cells were sequenced as 100 X 2 paired-end reads
   on an Illumina HiSeq 4000 using the HiSeq 3000/4000 sequencing kit and
   HD 3.4.0.38 collection software. Base-calling was performed using
   Illumina’s RTA version 2.7.7. In order to validate the transcriptomic
   PCR data, an independent set of experiments were performed using
   conventional real-time qPCR and were confirmatory (Supplemental
   Figure [63]3).

Bioinformatics analysis

   Gene expression counts were obtained using the MAP-RSeq v.1.2.1.5
   workflow [[64]21], the Mayo Bioinformatics Core pipeline. MAP-RSeq
   consists of alignment with TopHat 2.0.6 [[65]22] against the human hg19
   genome build and gene counts with the HTSeq software
   ([66]http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html).
   Gene annotation files were obtained from Illumina
   ([67]https://support.illumina.com/sequencing/sequencing_software/igenom
   e.html). Normalized gene counts were obtained from MAP-RSeq, where
   expression levels for each gene were normalized to one million reads
   and corrected for gene length (fragments per kilobase pair per million
   mapped reads [FPKM]). For heatmap and principal component analysis
   (PCA) visualizations, significant genes were selected based on an
   average FPKM > 1, a log-fold-change >or < absolute value of 1, and a p
   value < 0.05, when comparing aCSF-treated to PLF media-treated MSCs.
   Gene expression values (FPKM) were log transformed and z-scaled by gene
   prior to plotting. Visualizations were constructed in R using the
   ComplexHeatmap [[68]23] and factoextra
   ([69]https://github.com/kassambara/factoextra) packages. A heatmap of
   genes expressed at FPKM > 1 on average and significantly up- or
   down-regulated (fold change > 2 or < 0.5, p < 0.05) between
   aCSF-treated and PLF media-treated MSCs was generated using the Broad
   Institute’s Morpheus tool
   ([70]https://software.broadinstitute.org/morpheus). Functional
   annotation analysis was performed using DAVID Bioinformatics Resources
   6.8 [[71]24, [72]25]. Pathway enrichment analysis of MSCs cultured in
   aCSF and CM was completed using GSEA software (version 4.1.0) [[73]26,
   [74]27]. Human_MSigdb_January_13_2021_symbol.gmt and
   Human_GOALL_no_GO_iea_January_13_2021_symbol.gmt from
   [[75]http://baderlab.org/Genesets] enabled identification of canonical
   pathways (hallmark gene sets, BIOCARTA, PID Pathways) and GO gene sets
   that were enriched in aCSF-treated MSCs [[76]28]. Gene sets with
   FDR < 0.25 (canonical pathways) or FDR < 0.1 (GO) were considered
   significant.

VEGF ELISA

   To measure the concentration of VEGF protein secreted in vitro from
   patient adMSCs, cells were again seeded at a density of 3000 cells/cm^2
   in triplicate, and the media was changed upon reaching ~ 80% confluence
   to fresh CM, PLF, or aCSF media. The supernatant media was collected
   and the underlying monolayer was harvested at 24 or 48 h time points.
   Media and cell pellets were flash frozen on dry ice and stored at -80C
   for DNA or RNA isolation. VEGF within supernatant media was measured
   using the Quantikine ELISA kit (R&D Systems, Minneapolis MN USA)
   according to the manufacturer’s instructions using a two-parameter
   logistical standard curve. VEGF levels were normalized to total DNA
   following extraction using DNEasy Blood & Tissue Kit (Qiagen, Valencia,
   CA, USA) and nanodrop quantification.

Measurement of analyte concentrations in human CSF

   ALS patients enrolled in a previously completed clinical trial
   ([77]ClinicalTrials.gov #[78]NCT01609283) received a dose of autologous
   adipose-derived MSCs, and CSF was obtained before treatment and 1 week
   after treatment for analysis [[79]17]. Analytes in CSF were measured in
   duplicate using commercially available immunoassays from Meso Scale
   Discovery according to their protocols. Samples were diluted with
   assay-specific diluents using recommended dilutions for CSF. If no
   recommendation was provided, the appropriate dilution was determined
   through linearity of dilution tests. MMP-1 and GDF-15 concentrations
   were measured with R-PLEX assays using CSF diluted 1 in 10 or 1 in 50,
   respectively. MCP-1 concentrations were measured with singleplex V-PLEX
   assays using CSF diluted 1 in 16. VEGF, PIGF, and bFGF concentrations
   were measured using a multiplex V-PLEX assay using CSF diluted 1 in 2.
   Each assay plate contained interplate control samples; interplate
   coefficient of variations for each assay type ranged from 1.79 to
   8.84%. Response values corresponding to the intensity of emitted light
   upon electrochemical stimulation of the assay plate using the Meso
   Scale Discovery QUICKPLEX SQ120 were acquired, and analyte
   concentrations were interpolated using MSD Discovery Workbench
   software.

Statistical analysis

   GraphPad Prism software was used for statistical analysis. Where
   appropriate, non-parametric data were analyzed by the Kruskal-Wallis
   test with Dunn’s test to correct for multiple comparisons.

Results

MSCs cultured in 100% human CSF for 24 or 48 h remain morphologically
unchanged

   To analyze the morphological changes of adipose-derived MSCs in CSF,
   all four lines of MSCs were cultured in CM, PLF media, or hCSF for 24
   or 48 h. At each time point, cells were stained with DAPI, phalloidin,
   and MitoTracker Red CMX Ros to image the nucleus, cytoskeleton, and
   mitochondria distribution. All of the surviving cells in each condition
   showed a fibroid-like morphology, with cells spread out in flat
   monolayers (Fig. [80]1a). Cells displayed branched cytoplasms and
   intact cytoskeletons. Mitochondrial distribution was normal, with most
   mitochondria being concentrated around the nuclei and filopodia. Nuclei
   appeared elliptical and speckled as expected. Overall, we detected no
   alterations in cellular morphology between cells cultured in each of
   the three conditions.

Fig. 1.

   [81]Fig. 1
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   Morphology and viability of MSCs cultured in CM, PLF media, and human
   or artificial CSF. a Morphology of MSCs imaged after 24 h in CM, PLF
   media, or hCSF. Cells were stained with phalloidin AF488 (green), DAPI
   (blue), and MitoTracker CMX-Ros (red). Images were taken at 40x
   magnification. b Metabolic activity of MSCs via MTS assay after 24–96 h
   in CM. c Metabolic activity of MSCs via MTS assay after 24–96 h in PLF
   media. d Metabolic activity of MSCs via MTS assay after 24–96 h in aCSF
   (n = 3, mean ± standard deviation)

MSCs exhibit decreased metabolic activity in PLF media and aCSF

   We cultured the four lines of MSCs in CM, PLF media, or aCSF for
   24–96 h to assess cellular proliferation and compare metabolic activity
   over time. As expected, cells in CM showed a normal increase in
   cellular activity, denoting a normal proliferation rate (Fig. [83]1b).
   In contrast, cells in nutrient-deprived PLF and aCSF showed a
   progressive decrease in metabolic activity, most likely due to
   decreased proliferation and viability (Fig. [84]1c, d). Rates of
   metabolic activity loss varied between cell lines, and it is unclear
   whether the cells remaining after 96 h in aCSF had undergone
   replication during that time.

Consistent transcriptomic patterns are dependent on culture condition

   To determine how the transcriptomes of MSCs change in response to CSF,
   we examined gene expression profiles in the four lines of MSCs cultured
   in CM, PLF media, and aCSF for 24 or 48 h using high-throughput RNASeq
   analysis. Unbiased transcriptomic analysis revealed homogeneity of both
   biological and technical replicates of MSCs cultured in CM, PLF media,
   or aCSF. Hierarchical clustering produced well-defined clusters of
   replicates, with the first order of separation being dictated by the
   treatment group and then by time in each condition (Fig. [85]2a).
   Principal components analysis identified culture condition and the time
   in each condition as accounting for over 50% of the variability between
   MSCs cultured in each condition (Fig. [86]2b). The number of annotated
   genes mapped from RNA reads totaled 23,398 for all conditions. Of these
   genes, in all conditions almost half of the genes were expressed at
   levels greater than 1 FPKM, about 10% were not appreciably expressed,
   and almost 50% were expressed at levels less than 1 FPKM (Fig. [87]2c).

Fig. 2.

   [88]Fig. 2
   [89]Open in a new tab

   Unbiased transcriptomic analysis reveals homogeneity of biological and
   technical replicates of MSCs treated with CM, PLF media, or aCSF for
   24–48 h. a Heatmap of results from samples submitted to RNA sequencing
   with well-defined clusters of replicates, with the first and second
   order of separation being dictated by the treatment group and time in
   the condition. b Principal component analysis demonstrates that culture
   condition and time account for over 50% of the variability in MSC
   transcriptomes. c Breakdown of expression levels of annotated genes for
   each culture condition. d Significantly enriched canonical pathways
   based on upregulated genes in aCSF-treated MSCs compared to CM-treated
   MSCs (FDR < 0.25). e Topmost enriched GO gene sets based on upregulated
   genes in aCSF-treated MSCs compared to CM-treated MSCs (FDR < 0.1)

MSCs cultured in aCSF maintain gene expression of MSC cell surface markers

   MSCs cultured in aCSF express mRNAs for cell surface markers that have
   been well-defined for MSCs [[90]20, [91]29–[92]31]. Standard cell
   surface markers for MSCs include CD44, CD73/NT5E, CD90/THY1, CD105/ENG,
   and MHC class 1 molecules. MSCs cultured in aCSF exhibit robust
   expression of the genes encoding these markers, albeit at varying
   levels as compared to cells cultured in CM or PLF media (Fig. [93]3a).
   Conversely, MSCs cultured in aCSF demonstrate an absence of expression
   of genes for hematopoietic, stem cell, or endothelial markers (CD11B,
   CD14, CD31, CD45, CD253A, HLA-DRA) except for CD34, which was expressed
   at very low levels (data not shown).

Fig. 3.

   Fig. 3
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   Expression levels of detectable transcripts of genes encoding
   characteristic MSC markers by MSCs cultured in CM, PLF, and aCSF. a
   Detectable expression levels of transcripts encoding surface markers
   for MSCs (n = 12, data shown with median and interquartile range). b
   Expression levels of transcripts encoding markers of proliferation and
   differentiation of MSCs. Transcript expression levels are shown as
   Log[2]FPKM (n = 12, data shown with median and interquartile range)

MSCs cultured in aCSF decrease expression of genes involved in proliferation
and increase expression of genes involved in promoting the non-canonical Wnt
signaling pathway

   As recommended by the IFATS/ISCT panel [[95]29], we examined expression
   of genes encoding factors involved in viability and fibroblastoid
   colony-forming units (CFU-Fs). As compared to cells in CM, cells
   cultured in PLF media and aCSF do not exhibit expression of MKI67,
   which encodes the proliferation marker Ki67, but retain expression of
   PCNA (Fig. [96]3b). We also found variable expression of extracellular
   matrix-associated genes that are reported to be up-regulated in
   post-proliferative cells (COL21A1, MGP, OGN, PODN), suggesting that
   cells incubated in aCSF are in a unique post-proliferative state
   [[97]20] (Fig. [98]3b).

   MSCs cultured in all three conditions expressed mRNAs for genes
   encoding reprogramming factors (MYC, KLF4, and KDM5B) to varying
   degrees (Fig. [99]3b). None of the cells cultured expressed genes for
   markers of pluripotency (e.g., NANOG, POU5F1, SOX2, LIN28A/B),
   corroborating previous observations [[100]20, [101]32]. Likewise,
   assessment of expression of lineage-specific genes revealed low-level
   expression of few genes for adipogenesis (PPARG), chondrogenesis
   (SOX9), and osteogenesis (ALPL, RUNX2), with these genes hypothesized
   to be playing an alternative role in driving matrix mineralization and
   cell survival in a post-proliferative state (Supp. Figure [102]1).

   Human primary cell quiescence, in addition to being signaled by cell
   cycle exit, is also promoted by suppression of the canonical Wnt
   signaling pathway [[103]33]. Notably, cells incubated in aCSF showed
   marked increases in genes associated with suppressing the canonical Wnt
   pathway (LRP4, RAPGEF1, AXIN1, DKK1, DKK3, TLE1-4) and a decrease in
   WNT2 expression (Supp. Fig. [104]2). Interestingly, aCSF-treated cells
   retained expression of genes encoding Wnt5A, Wnt5B, Wnt7B, and Wnt9A,
   which activate the non-canonical Wnt pathway to promote osteoblast
   differentiation and influence synaptic plasticity, which could impact
   the regenerative effects of MSCs in the CNS [[105]34, [106]35].

Expression of genes for secreted factors suggests the response by MSCs to
aCSF is immunomodulatory and enriched for growth factors

   It is important to assess changes in the expression of genes encoding
   secreted factors by MSCs in response to aCSF. Secreted factors
   potentially could serve as biomarkers of MSC activity in vivo, and the
   expression of genes for secreted growth factors specifically have
   mechanistic relevance to clinical trials that likely depend on the
   trophic functions of secreted factors.

   In examining the expression of immune-related cytokines, we found
   elevated expression of several factors in aCSF-treated MSCs as compared
   to PLF media-treated MSCs, including IL6 and CCL2 (encodes MCP-1) to
   levels comparable to CM-treated cells. MSCs lacked expression of
   pro-inflammatory factors. No expression was seen of TNFα, IFNγ, or IL33
   in any MSC culture condition, and IL32 was reduced in aCSF
   (Fig. [107]4a; non-expressed factors not shown).

Fig. 4.

   [108]Fig. 4
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   Expression levels of detectable transcripts of genes encoding key
   immunomodulatory, angiogenic, and neurotrophic factors by MSCs cultured
   in CM, PLF, and aCSF. a Detectable expression levels (FPKM) of genes
   encoding immunosuppressive and inflammatory factors (n = 12). b
   Expression levels (FPKM) of genes encoding select angioneurins and
   growth factors that are highly expressed by aCSF-treated MSCs (n = 12).
   c In vitro levels of VEGF protein in MSC cultures in CM, PLF, and aCSF.
   Data presented with mean ± standard deviation. The Kruskal-Wallis test
   with Dunn’s test to correct for multiple comparisons were performed to
   determine statistical significance (* indicates p < 0.05; ** indicates
   p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001)

   When we looked at the 281 and 392 genes that are significantly
   upregulated by aCSF-treated MSCs compared with PLF media-treated and
   CM-treated MSCs, respectively, we identified genes for several growth
   factors, including BDNF, FGF1, FGF2, GDNF, NGF, PGF, VEGFA, and VEGFB
   (Fig. [110]4b). Many of these factors promote angiogenesis, which was
   identified by functional annotation analysis as a top category of genes
   up-regulated in aCSF. Several of these factors are identified as
   angioneurins that have an effect on both angiogenesis and
   neuroprotection, such as VEGFA, NGF, and FGF [[111]36].

   ELISA assays were performed for VEGF on a separate set of MSC cultures
   exposed to CM, PLF, and aCSF. MSCs exposed to PLF had reduction in VEGF
   secretion when compared with CM. When exposed to aCSF, there was an
   initial drop in VEGF secretion at 24 h in culture, which recovered at
   48 h (Fig. [112]4c).

CSF growth factor protein levels are increased in humans after intrathecal
MSC injection

   To further assess MSC behavior in CSF, protein quantitation was
   performed on CSF samples from subjects with ALS before and after
   intrathecal MSC administration. Subjects received an intrathecal dose
   of 1 × 10^7, 5 × 10^7, or 10 × 10^7 autologous adipose-derived MSCs.
   The fold change in protein level comparing the 1 week time point to
   baseline levels was calculated for each patient and used for
   statistical analysis. A dose-dependent response trend was observed in
   VEGF protein levels for each group, with a 1.2 ± 0.14 fold change with
   1 × 10^7 MSCs, 3.8 ± 2.9 fold change with 5 × 10^7 MSCs, and a
   significant 25.3 ± 18.1 fold change with 10 × 10^7 MSCs (p = 0.006;
   Fig. [113]5). For PIGF and GDF-15, the 10 × 10^7 MSC dose again
   correlates with a significant increase in protein levels compared to
   the 1 × 10^7 and 5 × 10^7 doses (p ≤ 0.03 for all; Fig. [114]5).
   Statistical analysis demonstrated that the 5 × 10^7 dose did not
   correlate with significantly increased growth factor protein expression
   compared to the 1 × 10^7 dose for VEGF, PIGF, or GDF-15. There was a
   trend for dose-dependent increases in MCP-1 and MMP-1, but this did not
   reach statistical significance. Altogether, high-dose intrathecal MSC
   injections resulted in increased levels of growth factors in the CNS
   compartment measured 1 week after treatment.

Fig. 5.

   [115]Fig. 5
   [116]Open in a new tab

   Fold change in protein level at 1 week post-intrathecal injections of
   MSCs. Patients with ALS received an intrathecal injection of either
   1 × 10^7, 5 × 10^7, or 10 × 10^7 autologous adipose-derived MSCs
   (n = 4, 10, and 5, respectively), and CSF samples were collected before
   MSC injection and at 1 week post-injection. The fold change of each
   factor was calculated for each patient, and the mean fold change ±
   standard deviations are plotted. The Kruskal-Wallis test with Dunn’s
   test to correct for multiple comparisons were performed to determine
   statistical significance (* indicates p < 0.05; ** indicates p < 0.01)

Discussion

   Our work expands the growing body of knowledge regarding the response
   characteristics of MSCs to novel stimuli or challenging environmental
   conditions, which, in turn, informs the use of MSCs in clinical
   applications [[117]37]. While previous human in vivo studies have
   provided early evidence of therapeutic benefits in ALS following
   intrathecal MSC treatment [[118]17, [119]38, [120]39], the long-term
   viability and mechanism of action of MSCs in the CSF environment remain
   unclear [[121]40–[122]42]. To determine how MSCs fare in the
   intrathecal space, we conducted a comprehensive investigation of the
   effects of CSF on human adipose-derived MSCs (manufactured with
   GMP-grade reagents) that examined changes in morphology, metabolism,
   and gene expression. Despite no clear gross morphological changes in
   MSCs cultured in CSF, there was a significant reduction in metabolic
   activity and a dramatically altered gene expression profile.
   Transcriptomic analyses revealed that MSCs increase expression of genes
   for growth factors and immune-modulating factors following culture in
   CSF. VEGF secretion was likewise maintained in aCSF. Remarkably, the
   transcriptomic analysis mirrored the protein changes in CSF from
   subjects with ALS that were treated with intrathecal MSCs.

   The unbiased transcriptomic analysis also revealed that MSCs
   demonstrate a distinct, robust profile of gene expression when cultured
   in aCSF, PLF media, or CM. MSCs in aCSF and PLF retain the expression
   of genes encoding cell surface markers and reprogramming factors
   characteristic of MSCs in CM [[123]20, [124]43], but lack expression of
   markers of active cellular proliferation. However, mRNA levels for
   markers of post-proliferative cells were differentially expressed to
   varying degrees in aCSF-treated MSCs as compared to PLF media-treated
   and CM-treated cells. The differential expression of Wnt ligands and
   pathway modulators suggests that aCSF-treated cells are quiescent, yet
   retain the ability to differentiate and promote synaptic plasticity, in
   part due to the expression of factors along the non-canonical Wnt
   pathway and the conserved expression of reprogramming factors [[125]34,
   [126]35]. This expression pattern likely reflects that aCSF-treated
   MSCs—which decrease proliferation while sustaining trophic factor
   expression—are in a post-proliferative state that is distinct from that
   of hPL-deprived MSCs cultured in PLF media or confluent cells in CM.

   When we examined genes that were significantly upregulated in aCSF
   compared to PLF media and CM, numerous genes for immune-modulating
   factors that are expressed at more than twice the levels seen in PLF
   media were identified. The expression of anti-inflammatory factors and
   lack of expression of pro-inflammatory genes by MSCs in aCSF suggests
   that these cells would suppress, rather than promote, inflammation in
   the CNS, consistent with previous reports suggesting this
   anti-inflammatory effect is therapeutic in disease models [[127]41,
   [128]44]. Immune system abnormalities [[129]45–[130]47] and
   neuroinflammation [[131]48, [132]49] are increasingly recognized as
   contributors to disease pathology in ALS and are being explored as
   therapeutic targets in clinical trials.

   Strikingly, aCSF exposure augmented the expression of angiogenic and
   growth factor genes over and above expression levels seen in PLF media.
   Previous studies have reported that human MSCs, even under serum
   deprivation conditions, are highly angiogenic [[133]50–[134]53], and
   express growth factors that promote neuroprotection, remyelination, and
   therapeutic effects in animal models of neurological diseases
   [[135]54–[136]57]. In support of these prior studies, we found that
   cells incubated in aCSF express genes for angiogenic and growth factors
   (e.g., GDNF, MCP-1) at levels seen in CM-treated MSCs. Increased MCP-1
   and GDNF expression by MSCs delivered intrathecally or intramuscularly
   into a rat model of ALS has been shown to delay motor function loss and
   to increase survival [[137]41, [138]56]. In addition, this study found
   that aCSF caused expression of several genes for growth factors above
   the expression levels seen in CM (BDNF, FGF2, NGF, PGF, VEGFA, VEGFB).

   This study identified that MSCs upregulate VEGF expression in both in
   vitro and in vivo conditions, giving insight to the probable mechanism
   of action of MSCs. Notably, upregulation of VEGF protein can regulate
   both angiogenesis and neurodegeneration, including degeneration of
   motoneurons [[139]36]. For instance, studies have found that mice had
   reduced VEGF levels prior to developing symptoms of spinal and bulbar
   muscular atrophy and adult-onset motoneuron degeneration that resembled
   the degeneration seen in ALS [[140]58, [141]59]. Conversely,
   overexpression of VEGF improves motor performance and prolongs survival
   in rodent models of ALS, particularly when paired with enhanced GDNF
   expression [[142]60]. Likewise, expression of VEGF promotes
   neurogenesis and neural stem cell differentiation post-injury [[143]61,
   [144]62]. By increasing neural perfusion around sites of
   neurodegeneration and ischemia, VEGF could promote neuroprotection for
   surviving motoneurons in the spinal cord and stimulate
   neuroregeneration. This hypothesis is further supported by the early
   success of clinical trials using MSCs with enhanced VEGF secretion to
   treat neurodegenerative disorders [[145]11, [146]38]. We measured a
   dose-dependent increase in CSF VEGF in patients with ALS following
   intrathecal treatment of autologous adipose-derived MSCs. The
   combination of increased gene expression in in vitro conditions and
   increased protein levels in in vivo conditions support the claim that
   MSCs within the intrathecal space produce significant quantities of
   therapeutic growth factors. Alternatively, MSCs could induce CNS cells
   to upregulate growth factor production. Regardless of the source of
   growth factors, their increased quantities confirm that MSCs are
   exerting therapeutic effects within the CNS compartment. Furthermore,
   high doses of MSCs (10 × 10^7) were required to elicit significant
   increases in protein expression of beneficial growth factors. These
   data are critical for researchers using MSC-based treatments as there
   are very few studies comparing MSC doses on therapeutic effect
   [[147]16, [148]17].

   There are now several completed human clinical trials that have
   utilized intrathecal autologous MSC therapy for ALS [[149]17, [150]38,
   [151]39, [152]63]. While there are signs of efficacy of MSC treatment
   in ALS, it is also becoming clear that some patients are more or less
   likely to respond to this therapy. Understanding this variability in
   response to MSC therapy will be critical for understanding ALS
   pathogenesis, MSC therapies, and how best to translate this therapy to
   clinical practice. Berry et al. reported that higher CSF VEGF was
   correlated with lower CSF MCP-1 in patients treated with modified
   bone-marrow-derived MSCs [[153]38]. They also reported that lower
   post-MSC treatment MCP-1 correlated with better outcomes, which was
   also seen in Oh et al. [[154]39]. Our phase I study did not have
   clinical outcomes to correlate with the CSF findings; however, we are
   now pursuing these hypotheses as part of an active phase II clinical
   trial of MSCs in ALS (ClinicalTrials #[155]NCT03268603).

   To our knowledge, this is the first reported large-scale transcriptomic
   analysis of multiple lines of human adipose-derived MSCs exposed to
   undiluted aCSF or hCSF. Unbiased examination of the differential
   expression of over 23,000 genes provided the most detailed
   understanding to date of how the transcriptomic profile of
   clinical-grade MSCs varies between normal, hPL-deprived, and
   intrathecal-like culture conditions. By understanding how the response
   of MSCs to CSF resembles—and is distinct from—the response to CM or PLF
   media, the field gains an appreciation for the scope of the phenotypic
   responses of MSCs. We further have shown that intrathecal injection of
   adipose-derived autologous MSCs in subjects with ALS leads to a
   dose-dependent increase in CSF growth factors. From a therapeutic
   standpoint, if MSCs respond to individual culture conditions with
   unique gene expression profiles, then proper preparation of MSCs could
   impact the secretion of therapeutic factors and biomarkers pertinent to
   the response of patients to MSC treatment.

Conclusion

   Our results demonstrate that MSCs can remain viable and morphologically
   normal in CSF. Under intrathecal conditions, the cells will decrease
   metabolic activity and will alter their gene expression to adapt to the
   novel environment while maintaining high-level expression of supportive
   factors that can be measured in the CSF of subjects treated with
   intrathecal MSCs for ALS. Overall, our results identify potential modes
   of therapeutic efficacy as well as candidate biomarkers of MSC potency
   to guide clinical translation of MSC-based therapies for
   neurodegenerative diseases.

   This work validates the continued use MSCs in clinical trials, and also
   supports using the 10 × 10^7 dose to maximize therapeutic benefit.

Supplementary Information

   [156]13287_2021_2241_MOESM1_ESM.jpg^ (998.7KB, jpg)

   Additional file 1: Supplemental Figure 1. Expression levels of
   detectable transcripts of lineage-specific genes for adipogenesis,
   chondrogenesis, and osteogenesis. Expression levels of transcripts
   encoding markers of MSC lineage differentiation (n = 12, data shown
   with median and interquartile range).
   [157]13287_2021_2241_MOESM2_ESM.jpg^ (1.4MB, jpg)

   Additional file 2: Supplemental Figure 2. Expression levels of
   detectable transcripts of genes associated with the canonical and
   non-canonical Wnt signaling pathways. Expression levels of transcripts
   encoding markers of Wnt pathway signaling (n = 12, data shown with
   median and interquartile range).
   [158]13287_2021_2241_MOESM3_ESM.jpg^ (1.1MB, jpg)

   Additional file 3: Supplemental Figure 3. Expression levels of selected
   genes using real-time qPCR confirms transcriptonic dataset.

Acknowledgements