CNS fibroblast-like cells in stroke and other neurological disorders

Abstract

Fibroblasts are the most common cell type of connective tissues. In the CNS, fibroblast-like cells are mainly located in the meninges and perivascular Virchow-Robin space. The origins of these fibroblast-like cells and their functions in both CNS development and pathological conditions remain largely unknown. In this review, we first introduce the anatomical location and molecular markers of CNS fibroblast-like cells. Next, the functions of fibroblast-like cells in CNS development and neurological disorders, including stroke, CNS traumatic injuries, and other neurological diseases, are discussed. Third, current challenges and future directions in the field are summarized. We hope to provide a synthetic review that stimulates future research on CNS fibroblast-like cells.

Introduction

Fibroblasts are mesenchymal cells that exist in the interstitial space of all organs.¹ They have flattened nuclei and thin cytoplasm with tenuous processes. As the most common cell type of connective tissues, fibroblasts play important roles in structure support, injury repair, and fibrosis.^{2, 3} Our knowledge on fibroblast functions mainly comes from skin fibroblasts. Recent studies demonstrate that fibroblast-like cells also exist in the meninges and perivascular Virchow-Robin space, and exert important functions both during central nervous system (CNS) development and in pathological conditions.^{4, 5} There are, however, controversial findings on the source and function of fibroblast-like cells after CNS injury. For example, it is unclear whether meningeal or perivascular fibroblast-like cells accumulate at injury site after CNS injury. In addition, it is in debate whether fibroblast-like cells play a neuroprotective and detrimental role after CNS injury. In this review, we first introduce the anatomical location and molecular markers of CNS fibroblast-like cells. Next, the functions of fibroblast-like cells in CNS development and neurological disorders, including stroke, CNS traumatic injuries, and other neurological diseases, are discussed. Third, current challenges and future directions in the field are summarized. We hope to provide a synthetic review that stimulates future research on CNS fibroblast-like cells.

Location of CNS fibroblast-like cells

In the CNS, fibroblast-like cells are found in the meninges and perivascular Virchow-Robin space in both mice and humans.^5–7 Meninges, which cover the brain and spinal cord, are an important connective tissue derived from the neural crest and mesoderm. Neural crest-derived cells form meninges of the forebrain, while mesoderm-derived cells form meninges of the midbrain and hindbrain. Meninges differentiate into dura (outmost layer), arachnoid, and pia (innermost layer) starting at ~E13 in mouse.^{8, 9} Dura is a collagenous membrane containing loosely packed dural fibroblast-like cells, arachnoid is a translucent sheet containing closely packed arachnoid fibroblast-like cells joined by tight junctions and desmosomes, and pia consists of a thin layer of pial fibroblast-like cells mainly joined by gap junctions^10–12 (Figure 1). Between arachnoid and pia is the cerebrospinal fluid-containing subarachnoid space (SAS).^{8, 13} Trabeculae, collagen fiber cores coated with meningothelial cells (fibroblast-like cells), divide the SAS into compartments^10–12 (Figure 1). The SAS also contains blood vessels, which are covered by meningothelial cells.^{10, 12} Together with penetrating blood vessels, pia also runs into brain parenchyma. Therefore, fibroblast-like cells are also found in perivascular Virchow-Robin space, sandwiched between vascular smooth muscle cells and astrocytes endfeet^{5, 6, 10, 14–16} (Figure 1). These perivascular fibroblasts are exclusively found in large blood vessels, including arteries, arterioles, veins and venules. The various subtypes of fibroblast-like cells reflect a complex system, highlighting important and diverse functions of these cells in the CNS.

Figure 1.

Diagram illustration of meningeal and perivascular fibroblast-like cells. SAS, subarachnoid space.

Based on the anatomical distribution of fibroblast-like cells, it is speculated that they regulate brain influx/efflux function via the glymphatic system, a specialized fluid-transporting system that functions predominantly during sleep.¹⁷ Cerebrospinal fluid from the SAS flows into brain parenchyma via the perivascular Virchow-Robin space, where it crosses the glia limitans in an aquaporin-4-dependent manner.^18–21 In the interstitium, cerebrospinal fluid mixes with interstitial fluid and leaves the brain via perivenous space and along cranial and spinal nerves.^18–21 Although how exactly fibroblast-like cells affect the glymphatic system remains unknown, there are a few interesting hypotheses. For example, fibroblast-like cells may modulate glymphatic function via regulating aquaporin-4 expression on astrocytic endfeet. Alternatively, fibroblast-like cells may directly affect fluid-transporting conduits---the basement membrane by up-/down-regulating numerous extracellular matrix (ECM) proteins. Additionally, fibroblast-like cells may regulate the glymphatic system via acting on mural cells and/or astrocytes. These hypotheses should be tested in future research.

Markers of CNS fibroblast-like cells

Molecular characterization of fibroblasts is challenging. Although several markers, including retinaldehyde dehydrogenase type 2 (Raldh2),^{22, 23} fibroblast-specific protein 1 (FSP1),^{24, 25} vimentin,^{23, 26} platelet-derived growth factor receptor-alpha (PDGFRα),^{5, 15, 27} platelet-derived growth factor receptor-beta (PDGFRβ),²⁸ ER-TR7,^{6, 29–31} fibronectin,^{23, 32, 33} and collagen type I (Col1α1),^{5, 14, 22} have been used to identify fibroblasts, these markers have several major limitations. First, none of these markers are fibroblast-specific. For example, vimentin is also expressed in arachnoid/meningothelial cells, astrocytes and endothelial cells, in addition to fibroblasts.^{23, 26} PDGFRα labels both fibroblasts and oligodendrocyte progenitor cells (OPCs) in the CNS.^{5, 15, 27} PDGFRβ marks fibroblasts and mural cells.^{28, 34} Although FSP1 labels fibroblasts in physiological conditions, its expression is induced in hematopoietic cells, endothelial cells, and vascular smooth muscle cells after injury.^{24, 25} Second, many fibroblast markers are ECM proteins, whose expression is substantially upregulated after injury. Therefore, these markers do not always reflect fibroblast changes in pathological conditions. For instance, increased fibronectin expression does not necessarily mean more fibroblasts. Third, there are no universal or subtype/organ-specific fibroblast markers. All currently used fibroblast markers label mixed populations and their specificity for different subtypes of fibroblasts remains largely unknown. Col1α1 is a well-characterized fibroblast marker in the brain.^{5, 14, 22} However, it cannot distinguish dural/arachnoid/pial fibroblasts or discern meningeal fibroblasts from perivascular fibroblasts.⁵ In addition, ER-TR7 mainly labels meningeal fibroblasts, pericytes, and reticular fibers.^{6, 29–31} The advantages and disadvantages of commonly used markers for fibroblast-like cells are summarized in Table 1.

Table 1.

Comparison of commonly used markers for fibroblast-like cells

Fibroblast markers	Expression in other cell types	ECM protein	Comments	References
Raldh2	Oligodendrocytes, Astrocytes	No	Expressed in both meningeal and perivascular fibroblasts, but not specific for fibroblasts in the CNS. Upregulated after CNS injury.	22, 23
FSP1	Hematopoietic cells, Endothelial cells, Vascular smooth muscle cells	No	Expressed in both meningeal and perivascular fibroblasts, but not specific for fibroblasts in the CNS. Expressed outside the CNS.	24, 25
Fibronectin	Macrophages, Astrocytes	Yes	Expressed in both meningeal and perivascular fibroblasts, but not specific for fibroblasts in the CNS. Upregulated after CNS injury. Expressed in other cells after injury.	23, 32, 33
Vimentin	Arachnoid/meningothelial cells, Astrocytes, Pericyte, Endothelial cells	No	Expressed in both meningeal and perivascular fibroblasts, but not specific for fibroblasts in the CNS. Expressed outside the CNS.	23, 26
PDGFRα	Oligodendrocyte Precursor Cells	No	Expressed in both meningeal and perivascular fibroblasts, but not specific for fibroblasts in the CNS. Expressed outside the CNS.	5, 15, 27
ER-TR7	Pericytes, Reticular fibers	Yes	Predominantly expressed in meningeal fibroblasts and pericytes in the CNS. Expressed outside the CNS.	6, 29–31
Col1α1	Podocytes, Pericytes	Yes	Specifically expressed in meningeal and perivascular fibroblasts in the CNS. Upregulated after CNS injury. Expressed in pericyte and podocytes outside the CNS.	5, 14, 22
PDGFRβ	Mural cells, Some neurons	No	Expressed in both meningeal and perivascular fibroblasts, but not specific for fibroblasts in the CNS. Expressed outside the CNS.	28, 34

Together, these findings support the use of both anatomical location and multiple markers to identify fibroblasts. Future research should focus on identifying pan- and subtype/organ-specific fibroblast markers. These markers will open doors for new research and substantially move the field forward.

Fibroblast-like cells in CNS development

Expression studies suggest that meningeal fibroblast-like cells play important roles during brain development. Single-cell RNA sequencing analysis revealed distinct gene expression profiles of mouse fibroblasts in the pia, arachnoid, and dura at E14.5, suggesting different functions of these cells.¹⁶ Immunohistochemistry validated that arachnoid and pial markers are conserved between mouse and human fetal meninges.¹⁶ Meningeal fibroblast-like cells, in particular pial fibroblasts, are the main source of pial ECM proteins, including collagens and laminins, which serve as an attachment point for radial glial cells and physical barrier for migrating neurons.^{16, 35–37} Pial fibroblasts are also a major source of CXCL12, which regulates neuronal migration and positioning during development. In addition, fibroblast-like cells in the pia and arachnoid show enriched expression of retinoic pathway genes, indicating that these cells may contribute to the production of retinoic acid (RA), which induces the differentiation of neural progenitors into neurons in the cortex.^{16, 38}

Loss-of-function studies reveal an indispensable role of meningeal fibroblast-like cells in CNS development. Mice with hypomorphic mutations in Foxc1, a transcription factor mainly expressed in meningeal cells, show meningeal defects, cerebral basement membrane breakdown, cortical dysplasia, and neuronal over-migration during development.^{35, 39} Similarly, Foxc1-deficient mice exhibit reduced meningeal fibroblast-like cells and RA production during development, resulting in decreased neurons and severe CNS defects.^{16, 40} These Foxc1 knockout mice also display cerebrovascular growth defect and cerebral hemorrhages due to diminished meningeal RA and abnormal neocortical vasculature growth.⁴¹ Consistent with these findings, ablation of Raldh2, which mediates RA synthesis, leads to abnormal neurogenesis and cerebrovascular development in mice.⁴² In addition, mice lacking PDGF-C, a major ligand for PDGFRα, show abnormal meninges, neuronal over-migration in the cortex, and cerebral bleeding,¹⁵ possibly due to reduced PDGFRα⁺ meningeal fibroblast-like cells and decreased ECM proteins. Like Foxc1 mutant mice, humans with Foxc1 mutations develop similar developmental defects in the CNS.^{16, 43, 44} Together, these results highlight critical roles of meningeal fibroblast-like cells in brain development and meninges formation.

Unlike meningeal fibroblast-like cells, the function of perivascular fibroblast-like cells in brain development remains unknown. This is an important area that needs future research.

Fibroblast-like cells in neurological disorders

After CNS injury, fibroblast-like cells actively proliferate, migrate, and secrete excessive ECM proteins (e.g. collagen and fibronectin) and bioactive molecules (e.g. axon growth inhibitors), contributing to the formation of fibrotic scar. These changes are predominantly induced by transforming growth factor-β1 (TGF-β1) signaling, which promotes fibroblast activation and ECM protein expression.^{32, 45} Fibroblast-like cells and fibrotic scar exert both detrimental and neuroprotective roles depending on the timing after CNS injury. The process of CNS injury repair can be broadly categorized into three stages: inflammation, new tissue formation, and remodeling.^{3, 46} It has been shown that fibrotic scar generally inhibits neuronal regeneration in the remodeling stage due to the physical barrier and axon growth inhibitors in the scar.^{4, 47, 48} At the new tissue formation stage, however, fibrotic scar plays a beneficial role due to its effect on junctional integrity.⁴⁶ Here, we discuss the functional significance of fibroblast-like cells in various neurological disorders, including stroke, CNS traumatic injuries, and others. The origins and functions of fibroblast-like cells in stroke and CNS traumatic injuries are summarized in Figure 2.

Figure 2.

Origins and functions of fibroblast-like cells in stroke and CNS traumatic injuries. A, Schematic illustration of possible origins of fibroblast-like cells in CNS injury. Question marks indicate the exact origin of activated fibroblast-like cells remains unknown. B, The functions and potential mechanisms of fibroblast-like cells in stroke and CNS traumatic injuries.

Stroke

There is evidence suggesting that fibroblast-like cells migrate to injury site after stroke. For example, PDGFRβ⁺ and CD105⁺ stromal cells derived from large vessels but not capillaries proliferate, migrate to injury site, deposit ECM proteins, and contribute to scar formation after ischemic stroke in both mice and humans.⁴⁹ Since these cells do not express pericyte markers (NG2 and CD13), but show increased fibronectin expression,⁴⁹ it is believed that these PDGFRβ⁺ and CD105⁺ cells are fibroblast-like cells. Using Col1α1-GFP transgenic mice, it has been shown that Col1α1⁺ cells, which express multiple fibroblast markers and reside in the meninges and along large blood vessels in the brain, accumulate in stroke lesion.²²

Most studies support a neuroprotective role of fibroblast-like cells in stroke. First, Col1α1⁺ fibroblasts act as an important source of RA, which induces neural progenitor differentiation and promotes recovery in rodents.^{22, 50} Next, nestin⁺ cells with features of fibroblasts are found in ischemic core at day 14 but not day 3 after stroke in rats,⁵¹ suggesting that fibroblast-like cells may contribute to stroke recovery. Additionally, at the chronic stage in a mouse ischemic stroke model, Col1α1⁺ and fibronectin⁺ fibroblast-like cells in the peri-infarct regions strongly express periostin,^{52, 53} which induces proliferation/differentiation of neural stem cells and improves functional recovery in neonatal hypoxic-ischemic rats.⁵⁴ Furthermore, mice lacking AKAP12, which is heavily expressed in meninges and fibrotic scar, exhibit impaired fibrotic scar structure, increased leakage from lesions, extended inflammation, and aggravated tissue damage in the photothrombotic stroke model.⁴⁶

It should be noted, however, that there is also evidence suggesting that fibroblast-like cells play a detrimental role in stroke. Unlike in ischemic stroke, neutralization of periostin attenuates early brain injury, whereas administration of recombinant periostin exacerbates early brain injury after subarachnoid hemorrhage in mice,⁵⁵ In addition, activation of PDGFRα, which is expressed in fibroblast-like cells and OPCs, increases cerebrovascular permeability in both ischemic and hemorrhagic stroke in mice.^{56, 57} The relative contributions of fibroblast- and OPC-mediated PDGFRα activation in vascular damage after stroke, however, need future research.

These results suggest that fibroblast-like cells can be either beneficial or deleterious in stroke. One possible explanation for this disparity is that different subpopulations of fibroblast-like cells, including dural, arachnoid, pial, perivascular fibroblasts and meningothelial cells, may have distinct biological properties and functions in stroke. If this is true, the overall functions of fibroblast-like cells may depend on the location of injury. Specifically, if the injury predominantly occurs in deep brain regions without affecting meninges, the outcome is mainly determined by perivascular fibroblast-like cells. On the contrary, if the lesion occurs in superficial brain regions, where meninges are injured, both meningeal and perivascular fibroblast-like cells contribute to the outcome. It should be noted that we cannot exclude the possibility that fibroblast-like cells from one location may have both neuroprotective and detrimental functions. In this case, the overall outcome largely depends on the specific microenvironment rather than the location of injury. In addition, injury models and disease stages may also affect the functions of fibroblast-like cells. Future studies should elucidate the functions of different fibroblast populations in stroke and other neurological disorders.

A key question in the field is which subtypes of fibroblast-like cells participate in stroke pathogenesis. Controversial findings exist probably due to the lack of molecular markers to distinguish these cells. On one hand, there is evidence supporting that meningeal fibroblast-like cells contribute to stroke repair. Fibronectin⁺ and ET-TR7⁺ mouse meningeal cells have epithelial properties under normal condition.⁵⁸ After photothrombotic stroke, however, these cells show epithelial–mesenchymal transition and migrate into the lesion site,⁵⁸ indicating a pro-recovery role of meningeal fibroblast-like cells in stroke. However, it remains unclear whether these fibronectin⁺/ET-TR7⁺ meningeal cells are originated from dural, arachnoid, pial fibroblasts or meningothelial cells.

On the other hand, perivascular fibroblast-like cells have also been shown to regulate stroke pathogenesis. A time-course study reports that PDGFRβ is expressed only in blood vessel walls at day 3 after ischemic stroke and it co-localizes with both pericyte marker Desmin and fibroblast marker Col1α1 in mouse brains.³⁴ At day 7 after injury, however, PDGFRβ is detected in both blood vessel walls and nonvessel-associated fibroblast-like cells.³⁴ Although PDGFRβ co-localizes with both Desmin and Col1α1 in blood vessel walls, it only merges with Col1α1 in nonvessel-associated fibroblast-like cells.³⁴ These findings suggest that PDGFRβ⁺Col1α1⁺ fibroblasts in ischemic brains may come from blood vessel walls. The accumulation of PDGFRβ⁺Col1α1⁺ fibroblast-like cells in ischemic brain may be caused by proliferation of perivascular fibroblast-like cells and/or differentiation of pericytes. Although the underlying mechanisms remain largely unknown, there is evidence suggesting that PDGFRβ signaling may be involved. First, PDGFRβ^+/− mice exhibit enlarged infarct volume and decreased expression of fibroblast markers (fibronectin and Col1α1) after ischemic stroke.³⁴ Next, PDGFR (PDGFRα and PDGFRβ) signaling regulates pericyte proliferation and their differentiation into myofibroblasts in two mouse models of kidney fibrosis.⁵⁹ It is difficult to distinguish fibroblast-like cells and pericytes, especially after brain injury, due to the large number of molecular markers they share. Future research should focus on identifying markers that are able to differentiate these cells and addressing their functional significance in stroke pathogenesis.

CNS traumatic injuries

Fibroblast-like cells are identified at injury core in rodents after spinal cord injury (SCI) by electron microscopy.^{60, 61} Subsequent studies show that invading fibroblast-like cells express CTGF, a profibrotic and angiogenic protein, in rats after SCI.⁶² It has also been reported that fibroblast-like cells substantially upregulate fibronectin⁶³ and Col1α1⁶⁴ expression after SCI in rodents, suggesting that these cells contribute to fibrotic scar formation after SCI. In addition, fibroblast-like cells also contribute to glial scar formation after SCI. It has been shown that meningeal fibroblast-like cells enhance astrocyte reactivity and glial scar marker expression in an astrocyte-fibroblast co-culture system.⁶⁵ In a rat SCI model, ER-TR7⁺ and PDGFRβ⁺ meningeal fibroblast-like cells upregulate the expression of Col1α1, which induces glial scar formation via integrin-β1 receptor on astrocytes.⁶⁶ Like in SCI, fibroblast-like cells are also involved in the pathogenesis of traumatic brain injury (TBI). It has been shown that fibronectin⁺ fibroblast-like cells migrate to injury site after TBI and substantially upregulate TGF-β receptor, whose activation promotes fibroblast proliferation, ECM production, and fibrotic scar formation.³² This result indicates that fibroblast-like cells are a major target of TGF-β after TBI. In addition, ER-TR7⁺ fibroblast-like cells synthesize chondroitin sulfate proteoglycans, which strongly inhibit axon growth and regeneration, at the injury site after TBI in mice.⁶⁷

Functional studies support a detrimental role of fibroblast-like cells in SCI. First, inhibiting Col1α1-integrin-β1 signaling promotes functional recovery after SCI in mice.⁶⁸ Next, mice deficient in periostin, an ECM protein mainly expressed by scar-forming cells, display diminished PDGFRβ⁺ cell proliferation, reduced collagen expression, decreased fibrotic scar formation, and better functional recovery after SCI.⁶⁹ Consistent with this finding, administration of periostin neutralizing antibody leads to similar changes in mice after SCI.⁶⁹ Deferoxamine treatment, which inhibits the expression of ECM proteins and axon growth inhibitory molecules, reduces fibrotic scar formation and promotes axon regeneration in rats after SCI.⁷⁰ Suppression of fibrous scarring promotes neuronal and axonal regeneration, and induces functional recovery in a rat model of SCI.⁷¹ These results suggest that fibroblast-derived ECM proteins may be targeted in SCI therapies. In addition, knockdown of MiR-21–5p, which mediates TGF-β1-induced fibrosis-related gene expression, improves motor functional recovery in a mouse model of SCI.⁴⁵ Echoed with this report, microtubule stabilizing drugs, such as Epothilone B and Taxol, inhibit fibrotic scar formation and promote axon regeneration in rodent models of SCI by reducing TGF-β signaling.^{72, 73} These findings indicate that blocking TGF-β1 signaling may have a therapeutic effect in SCI. Furthermore, depletion of hematogenous macrophages results in decreased fibroblast-like cell number and increased axonal growth in a mouse model of SCI,⁷⁴ suggesting important roles of hematogenous macrophages in fibroblast-like cell recruitment and injury repair in SCI. Similarly, a detrimental role of fibroblast-like cell-derived ECM proteins is observed in TBI. For instance, suppressing fibrotic scar formation by inhibiting collagen type IV triple-helix formation promotes axonal regeneration without disturbing the healing process in TBI.⁷⁵ Additionally, local administration of chondroitinase-ABC, which degrades chondroitin sulfate proteoglycans, has been shown to significantly promote functional recovery in rats after SCI.⁷⁶ These findings suggest that inhibiting ECM deposition may be a reliable strategy to promote axonal regeneration after TBI.

Whether fibroblast-like cells at lesion sites are from meninges or blood vessels has been controversial and seems to depend on the types of injury. Using Col1α1-GFP transgenic mice, it has been demonstrated that Col1α1⁺ fibroblast-like cells predominantly come from blood vessels in the contusion model, in which the dura of meninges is intact.¹⁴ In contrast, there is also evidence suggesting that fibroblast-like cells migrate into injury sites from adjacent meninges in a rat spinal cord contusion model.⁷⁷ This discrepancy may be explained by different fibroblast markers used in these studies. In the penetrating SCI model that breaks the meninges, on the other hand, fibroblast-like cells seem to come from meninges rather than blood vessels.¹⁴ It is thus important to perform SCI research using both contusion and penetrating models. It is speculated that fibroblast-like cells predominantly come from meninges in TBI, which shares similar pathology as penetrating SCI injury. Future research should elucidate the origins of fibroblast-like cells in CNS traumatic injuries.

Other neurological disorders

Multiple sclerosis is an autoimmune disease. It has been reported that both PDGFRα⁺PDGFRβ⁺ meningeal fibroblast-like cells and PDGFRα⁻PDGFRβ⁺ pericytes increase and form a network in mouse meninges in the experimental autoimmune encephalitis (EAE) model of multiple sclerosis.⁷⁸ Upon inflammatory stimuli, such as TH17 cells and IL-17/IL-22, these cells secrete ECM proteins and cytokines, leading to enhanced immune response and demyelination.⁷⁸ These findings suggest that meningeal fibroblast-like cells may play a detrimental role in EAE. Using Col1α1-GFP transgenic mice, it has been shown that perivascular fibroblast-like cells in the spinal cord become activated and infiltrate into neural tissue in EAE.⁷⁹ Interestingly, this change is associated with animal behavioral deficits, demyelination, myeloid cell accumulation, and ECM deposition,⁷⁹ indicating a detrimental role of perivascular fibroblast-like cells in EAE. In addition, fibroblast-conditioned media and fibroblast-derived ECM have been found to inhibit OPC differentiation in vitro.⁷⁹ Given that abnormal ECM deposition is observed in multiple sclerosis in both mice⁸⁰ and humans,^{81, 82} it is believed that fibroblast-like cells affect multiple sclerosis pathology and outcomes at least partially via their ECM proteins. This hypothesis and the underlying molecular mechanisms, however, need further investigation.

Fibroblast-like cells also contribute to scar formation in 3-nitropropionic acid-induced Huntington’s disease model. It has been shown that collagen-producing PDGFRβ⁺ fibroblast-like cells increase and extend into extravascular space in the lesion core over time in 3-nitropropionic acid-treated rats.⁸³ Ultrastructural analysis demonstrates that these PDGFRβ⁺ fibroblast-like cells have close interaction with macrophages,⁸³ suggesting a possible role of macrophages in fibroblast migration and fibrotic scar formation. Biochemical analysis shows that these PDGFRβ⁺ fibroblast-like cells are induced to express nestin and vimentin in the lesion core,⁸³ highlighting important roles of nestin and vimentin in the structural dynamics of fibroblast-like cells. The functional significance of fibroblast-like cells in Huntington’s disease and other neurodegenerative disorders, however, remains unknown and needs future investigation.

Conclusions

Fibroblast-like cells are stromal cells with high plasticity. With the advance of imaging and next generation sequencing techniques, CNS fibroblast-like cells and their subpopulations have been identified. Several challenges, however, prevent the study of these cells. First, there are no pan- or subtype-specific fibroblast markers available currently, which makes isolation of pure fibroblast populations and investigation of their functions impossible. Second, fibroblast-like cells are highly plastic. They are able to differentiate and/or de-differentiate into other cell types after CNS injury, which makes it difficult to define and characterize their functions. Third, there are no genetic tools that allow for loss-of-function studies. The development of such tools (e.g. mice lacking a subpopulation of fibroblasts) relies on subpopulation-specific markers. Fourth, the origins of fibroblast-like cells in CNS injuries are largely unknown. It remains unknown if fibroblast-like cells at lesion sites are originated from dural, arachnoid, pial, perivascular fibroblasts and/or meningothelial cells (Figure 2). It seems the location and nature of injury may affect the types of fibroblast-like cells accumulated at lesion sites. Fifth, the functions of fibroblast-like cells in development and pathological conditions are largely unknown. Current knowledge on the functions of fibroblast-like cells mainly comes from association studies, in vitro experiments, and/or non-CNS fibroblasts. It remains unclear if different subtypes of fibroblast-like cells have distinct functions, and if one subtype of fibroblast-like cells can exert both beneficial and deleterious effects in CNS injuries. Fifth, the molecular mechanisms responsible for the migration and functions of different subtypes of fibroblast-like cells in physiological and pathological conditions are unclear. Answering these important questions will address key challenges and substantially move the field forward.

Non-standard Abbreviations and Acronyms:

CNS

Central nervous system

Col1α1

Collagen type I

ECM

Extracellular matrix

FSP1

Fibroblast specific protein 1

OPCs

Oligodendrocyte progenitor cells

PDGFRα

Platelet-derived growth factor receptor-alpha

PDGFRβ

Platelet-derived growth factor receptor-beta

Raldh2

Retinaldehyde dehydrogenase type 2

RA

Retinoic acid

SCI

Spinal cord injury

SAS

Subarachnoid space

TGF-β1

Transforming growth factor-β1

TBI

Traumatic brain injury