Classes of Stem Cells: From Biology to Engineering

Abstract

Purpose

The majority of adult tissues are limited in self-repair and regeneration due to their poor intrinsic regenerative capacity. It is widely recognized that stem cells are present in almost all adult tissues, but the natural regeneration in adult mammals is not sufficient to recover function after injury or disease. Historically, 3 classes of stem cells have been defined: embryonic stem cells (ESCs), adult mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs). Here, we have defined a fourth fully engineered class: the synthetic artificial stem cell (SASC). This review aims to discuss the applications of these stem cell classes in musculoskeletal regenerative engineering.

Method

We screened articles in PubMed and bibliographic search using a combination of keywords. Relevant and high-cited articles were chosen for inclusion in this narrative review.

Results

In this review, we discuss the different classes of stem cells that are biologically derived (ESCs and MSCs) or semi-engineered/engineered (iPSCs, SASC). We also discuss the applications of these stem cell classes in musculoskeletal regenerative engineering. We further summarize the advantages and disadvantages of using each of the classes and how they impact the clinical translation of these therapies.

Conclusion

Each class of stem cells has advantages and disadvantages in preclinical and clinical settings. We also propose the engineered SASC class as a potentially disease-modifying therapy that harnesses the paracrine action of biologically derived stem cells to mimic regenerative potential.

Lay Summary

The majority of adult tissues are limited in self-repair and regeneration, even though stem cells are present in almost all adult tissues. Historically, 3 classes of stem cells have been defined: embryonic stem cells (ESCs), adult mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs). Here, we have defined a fourth, fully engineered class: the synthetic artificial stem cell (SASC). In this review, we discuss the applications of each of these stem cell classes in musculoskeletal regenerative engineering. We further summarize the advantages and disadvantages of using each of these classes and how they impact the clinical translation of these therapies.

Introduction

The majority of adult tissues are limited in self-repair and regeneration due to their poor intrinsic regenerative capacity. The limitation of mammalian tissue regeneration and repair stems from the insufficient tissue-specific stem cell contribution as opposed to an abundant pluripotent reserve of stem cells in animals with high regeneration capacity. Additionally, mammalian cells have a limited capacity to dedifferentiate upon injury, but the dedifferentiation potential needs to be stimulated for regeneration [1]. Therefore, in mammals, disease or tissue damage often leads to irreversible functional failure in the body. Often, pharmacological agents have been used to slow disease/degeneration progression, but while these treat the symptoms, a disease-modifying therapy is highly sought after. It is widely recognized that stem cells are present in almost all adult tissues [2]. The ability of stem cells to self-renew, differentiate into multiple lineages, and release molecules to signal tissue repair makes them a primary choice for tissue engineering studies [2]. Even with the presence of stem cells, the natural regeneration in adult mammals is not sufficient to recover function after injury or disease, which leads us to develop a strategy to enhance our regeneration potency.

Historically, three classes of stem cells have been defined: embryonic stem cells (ESCs), adult mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs). Biologically derived stem cells have gained much attention in the regenerative engineering field due to their abundance throughout the body. Cells isolated from each source have different properties that can be used for engineering different tissues [2]. Genetic engineering approaches have been taken to reprogram differentiated cells into a pluripotent state (iPSCs). With the introduction of the iPSCs, tissue engineering has become more versatile with the ability to reprogram cells from abundant sources to make tissue where cells may not be as readily available [3]. However, stem cell therapy has still not been realistic for clinical translation for several reasons: (1) with high doses of stem cells required, a large expansion is needed [4]; (2) the stem cells change morphological, phenotypic, and genetic changes through each expansion, which can lead to loss of therapeutic potential [5]; (3) the lack of successful engraftment, migration into the injured site, loss of functionality and viability, and the possibility of transmission of infectious remain challenges associated with direct cell transplantation [6, 7]; and (4) less sources of stem cells would produce low cell yields, making expansion to the required cell dose more difficult [5, 8].

Here, we discuss the three classes currently used for regenerative engineering applications, and we also define a fourth class: the synthetic artificial stem cell (SASC). We start our review with two classes that are completely biologically derived classes (ESCs and MSCs) and extend into the semi-engineered class (iPSCs). Traditionally, the definition of a stem cell is its self-renewal ability and multilineage differentiation potency. We expand upon the definition of a stem cell with the introduction of a fully engineered class (SASC) that controls the delivery of paracrine factors like biological/semi-engineered stem cells but with the added ability to engineer modification of disease progression. We focus on tissue engineering applications for the treatment of musculoskeletal diseases and assess the advantages and challenges each class faces for clinical translation.

Biologically Derived Stem Cell Classes

Class 1: Embryonic Stem Cells

Embryonic stem cells (ESCs) were originally isolated from the inner cell mass of mouse blastocysts in the 1980s [9, 10]. Seventeen years later from the generation of mouse ESCs, human ESCs were first reported in 1998 [11]. ESCs possess infinite proliferation ability and are recognized as typical pluripotent stem cells that can differentiate into all cell types, whereas stem cells obtained from other tissues are multipotent or unipotent [12]. ESCs retain their self-renewal capability and pluripotency in the presence of anti-differentiation factors or are co-cultured with a layer of mouse embryonic fibroblasts [13]. It has been reported that mouse ESCs require leukemia inhibitory factor (LIF) and bone morphogenic proteins (BMPs) for self-renewal, whereas human ESCs require fibroblast growth factors (FGFs) and suppression of BMP signaling [14–17]. When ESCs are grown in suspension without those inhibitory factors, ESCs undergo spontaneous differentiation and form three-dimensional aggregates called embryoid bodies (EBs) that comprise the three embryonic germ layers (ectoderm, mesoderm, and endoderm) as well as the germline [18, 19]. Since EBs recapitulate various aspects of cell differentiation during embryogenesis [20], ESCs have been used to study the mechanism of in vitro differentiation into a variety of cell types (Fig. 1). Human ESCs also have served as in vitro primary cell models to screen the activity and toxicity of candidate compounds for drug discovery [21]. Drug discovery relies on the availability of reliable cell types and disease models. The function and effect of candidate drug compounds can be validated in ESC-derived-specific differentiated cell types. Furthermore, stem cells can be used to form three-dimensional self-organized tissue or organ models through cell sorting out and spatially restricted lineage commitment called organoids [22]. Various ESC-derived organoids, including the optic cup [23], brain [24], intestine [25], kidney [26], and limb mesenchyme [27], have been reported. ESC-derived organoids also can be used to model disease by introducing disease mutations and drug testing [22].

Fig. 1.

The use of embryonic stem cells (ESCs) for studying basic developmental biology, disease mechanisms, and treatment of disease

ESCs have been explored in vitro and preclinical animal studies for various diseases and injuries such as Parkinson’s disease, macular degeneration, spinal cord injury, myocardial infarction, and type I diabetes, and some of them are tested in clinical trials [28]. The feasibility of using human ESC (hESC) in a porcine osteoarthritis (OA) model has been investigated [29]. In this work, Petrigliano et al. showed that in a pig model of articular defect, chondrocytes derived from membrane-bound human ESC had improved repair of articular cartilage defects and superior biomechanical competency at 6 months. Furthermore, hESC has also been used for functional skeletal muscle regeneration [30]. Albini et al. demonstrated that in undifferentiated ESCs, forced expression of SWI/SNF component BAF60C (encoded by SMARCD3) enabled MyoD positioning and chromatin remodeling. The study conferred that BAF60C is the crucial epigenetic factor that determines the commitment of hESC to myogenic differentiation.

Although the capacity of ESCs to differentiate into various cell types is well documented, the transplantation of ESCs or ESC-derived cells for cell therapy is still not realistic. Initial criticism for ESC transplantation is the ethical concern of using human embryos. To overcome this, ESCs have been successfully generated by somatic cell nuclear transfer into the human oocyte [31]. However, this approach depends on the oocyte quality of the donor and remains technically challenging with low efficiency.

Moreover, several practical issues of ESC transplantation have been recognized, such as low engraftment rate and tumorigenicity. For example, transplantation of ESC-derived cardiovascular progenitor cells in the heart of primates after myocardial infarction resulted in less than 0.2% cell survival at 28 days post-transplantation despite immunosuppressive conditions [32]. On the other hand, ESC-derived hepatocytes produced human albumin in immunodeficient mice for up to 74 days [33], and ESC-derived endocrine cells produced insulin in immunodeficient mice for up to 94 days [34]. Integration of transplanted cells in host tissue is critical to recovering tissue function. Therefore, depending on the disease and injury models, an appropriate dose of transplanted cells and long-term engraftment needs to be carefully evaluated. Tumorigenicity is a classic disadvantageous characteristic of pluripotent stem cells. Several studies reported that transplantation of ESC-derived cells induced teratoma formation in various disease models [35, 36]. This risk can be controlled by using differentiated ESC and the cell sorting system to eliminate pluripotent stem cells [28, 37]. In addition, long-term follow-up is necessary to exclude the possibility of teratoma formation. The accumulation of fundamental knowledge of ESCs drives diverse clinical applications, but most clinical trials that use human ESCs are suspended in early phases [28, 38]. The potential of ESCs for cell therapy depends on overcoming obstacles, especially the quality of control, safety, and efficacy.

Class 2: Adult Mesenchymal Stem cells

Adult stem cells are groups of undifferentiated cells present in the body which have the ability to differentiate either into several lineages (multipotent) or restricted to a single-cell type (unipotent). The primary role of adult stem cells is to repair and self-renewal of tissues. Multipotent adult stem cells include hematopoietic, neuronal, intestinal, cardiac, and mesenchymal stem cells, whereas unipotent stem cells include satellite stem cells and epidermal stem cells [39]. In this review, we focus on the stem cells of musculoskeletal origin: adult mesenchymal stem cells (MSCs).

In the 1970s, Friedenstein et al. first reported the presence of a population of spindle-shaped, fibroblast-like, non-hematopoietic cells that had the potential to develop into fibroblastic colony-forming units [40]. This cell population, isolated from the stroma of bone marrow and spleen, could retain the ability to differentiate into adipocytes and osteoblasts [41]. After almost two decades, Caplan, in 1991, identified these cell populations in the periosteum and adult bone marrow as mesenchymal stromal/stem cells (MSCs), which could differentiate into osteoblasts and chondrocytes [42], and connective tissues of mesenchymal origin [43]. Since then, adult MSCs have been isolated from multiple tissue sources like the adipose tissue [44], Wharton’s jelly [45], umbilical cord [46], synovial fluid [47], skin [48], bone marrow [49], and several other tissues.

Traditionally, the isolation of MSCs is based on their capacity to adhere to plastic and easy expansion ex vivo [50]. MSCs are multipotent and can be differentiated into osteoblasts, chondrocytes, and adipocytes (Fig. 2). Additionally, according to the International Society of Cellular Therapy, MSCs should be positive for markers CD90 (Thy-1), CD105 (SH2), and CD73 (SH3/4) and should be negative for CD14, CD34, CD45, CD11b, CD19, or major histocompatibility complex class II (MHC-II) [51]. The two main sources of MSC isolation are bone marrow (bone marrow–derived stem cells/BMSCs) and adipose tissue (adipose tissue–derived stem cells/ADSCs). Although bone marrow is indicated as the primary source of MSCs, BMSCs account for roughly 0.001–0.01% of mononuclear cells [52]. Compared to BMSCs, ADSCs are easy to harvest, have abundant sources, and have a higher yield of stem cells (approximately 2% of nucleated cells) [53]. Following isolation and culture, MSCs are maintained by periodic passaging and confirmed by the presence or absence of MSC markers by flow cytometry.

Fig. 2.

Mesenchymal stem cells (MSCs) used for musculoskeletal applications have been primarily isolated from bone marrow and adipose tissue. Stem cells isolated from these tissues have a multipotent capacity to differentiate into osteogenic, chondrogenic, or adipogenic lineages. These cells have been transplanted into different musculoskeletal tissues either through direct injection or by loading into various biomaterials, which can be used to enhance cell integration

The ability of MSCs to self-renew and differentiate into multiple lineages is of primary advantage in the field of regeneration and repair. It is believed that chemical signaling and chemical gradients during tissue damage aid in MSC homing to the target sites mediated by chemokine receptors [54]. The functional repair at those sites and adequate differentiation in different cell lineages may depend on the stiffness of the tissues [55]. It has been reported that soft gels supported MSC differentiation to adipogenesis, while stiff substrates favored osteogenesis [55–57]. Thus, long-term MSC culture, either in a soft or stiff substrate, helps the cells to commit to a particular lineage by switching the substrates and soluble factors, although the underlying molecular basis is still unknown [58, 59].

MSCs also have the ability to secrete bioactive factors that signal tissue healing. This composition of factors, collectively known as the secretome, plays important roles in biological functions such as homeostasis, immunomodulation, anti-apoptosis, anti-inflammation, and angiogenesis [60]. Similar to the lineage fate of the MSCs, the composition of factors in the secretome is dynamic and dependent on the microenvironmental stimuli and various physical cues.

A wide range of clinical conditions intends to use adult MSCs for therapeutic purposes, as evidenced by the recent increase in clinical trials using MSCs (Fig. 2) [61]. One of the foremost advantages of MSCs for therapeutic applications is that they can be isolated from various tissue sources with the potential to differentiate into multiple lineages. MSCs are also attractive stem cell sources for transplantation owing to their immunosuppressive and anti-inflammatory roles which is advantageous since they can resist immune rejection in the host organism [62–64]. Thus, the low immunogenicity of the MSCs makes them well-tolerated post-transplantation in the recipients. Since MSCs can be easily obtained from the patient’s body like blood or adipose tissues, with minimally invasive surgeries, which can then be used for expansion, it makes it clinically feasible for large-scale clinical use. Furthermore, since the source of the stem cells is adult tissue, it does not raise ethical concerns as in ESCs [65]. Thus, adult MSCs have therapeutic advantages over other types of stem cells for clinical use. Clinical trials with adult stem cells, and in some cases therapeutic applications, have found their way into heart diseases, neurogenesis, stroke, spinal cord injury, liver disorder, diabetes, ophthalmic disorders, etc. [66–68].

The use of MSCs for osteoarthritis (OA) and osteoporosis has gained substantial attention and progress over the past decade. OA is a chronic articular cartilage degenerative disease along with subchondral bone remodeling, osteophyte formation, and inflammation [69]. Stem cell–based therapy has been extensively investigated at both preclinical and clinical stages [70]. Both bone marrow–derived (BMSCs) and adipose tissue–derived MSCs (ADSCs) are attractive sources for transplantation studies in OA. In a sheep model of OA, Al Faqeh et al. showed that groups injected with BMSCs could retard the OA progression with good cartilage thickness and histomorphometry [71]. In another study involving a mouse model of posttraumatic arthritis (PTA), intra-articular injections of BMSCs increased bone volume and prevented PTA, while the control group with saline developed PTA [72]. For cartilage regeneration in OA, adipose tissues are an attractive source since it is abundantly available in the body, and it is safer to harvest involving a less invasive procedure.

In the past decade, several clinical trials for ADSC-based cell therapies in OA have been conducted as an alternative and effective treatment for OA. In 2014, in a clinical trial involving 18 patients with a median age of 63, intra-articular injections of ADSCs derived from a patient’s abdominal fat (autologous) improved knee OA-associated pain and reduced cartilage defect size with improved thick hyaline cartilage-like regeneration [73]. Treatment-associated adverse effect was not reported in this study. Furthermore, in 2016, a biocentric uncontrolled phase 1 clinical trial was conducted in patients with severe knee OA. In this study, patients were given a single intra-articular injection of autologous ADSCs with three doses of low, medium, or high. Six months after follow-up, this procedure was deemed safe without any serious adverse side effects [74]. More recently, in 2019, a randomized, double-blinded, and controlled phase II clinical trial was conducted on 53 patients with symptomatic knee OA. Patients randomly received either ADSCs derived from the patient’s body, expanded and combined with cell suspension solution, or hyaluronic acid (HA). After 12 months, the ADSC group showed significant improvements in articular cartilage volume, pain, and cartilage regeneration without major adverse effects [75]. As of 2022, there are 20 clinical trials with BMSCs for knee OA (search option “knee osteo arthritis” and “bone marrow-derived stem cells”) and 39 clinical studies registered for OA studies using ADSCs (search option “knee osteo arthritis” and “Adipose-derived stem cells”) with only a few studies with completed outcomes as shown in Table 1.

Table 1.

Clinical study outcomes in knee OA patients using adult mesenchymal stem cell source

Adult MSCs	Clinical study design	Study outcome	Reference
BMSC	A single intra-articular injection of 1, 10, or 50 million cells was given to patients with late-stage Kellgren-Lawrence knee OA	No serious adverse events, significant improvement of KOOS pain, symptoms, and WOMAC stiffness compared to baseline, significantly lower cartilage catabolic biomarkers	[76]
BMSC	Intra-articular injection of hyaluronic acid alone (control) or together with 10 × 106 (low dose) or 100 × 106 (high dose) cultured autologous BM-MSCs were administered in OA patients’ knees in a randomized study	No serious adverse events, WOMAC and VAS values for pain improved in both low and high doses	[77]
BMSC	Intra-articular single injection of autologous stimulated BMSCs (subcutaneous administration of granulocyte colony-stimulating factor (G-CSF)) with a control group receiving oral acetaminophen	No adverse effect, the BMSC group significantly improved pain and quality of life after 6 months	[78]
BMSC	Single superolateral knee injection of 50 × 106 or 150 × 106 allogeneic BM-MSCs into OA knees 7–10 days after the meniscectomy, with a control group of sodium hyaluronate (hyaluronic acid/hyaluronan) vehicle	No ectopic tissue formation, no serious adverse effects, significantly increased meniscal volume, improved VAS pain scores	[79]
BMSC	Intra-articular injection of 40 × 106 autologous cells in patients with severe OA	Improved pain outcomes from VAS and WOMAC scores, improved cartilage quality as demonstrated by T2 relaxation measurements	[80]
ADSC	Intra-articular injection of autologous ADSCs with a single dose of 1 × 108 cells, randomized, double-blinded, and placebo-controlled study	No serious adverse outcomes, compared to the control (saline) groups, a single injection of ADSCs led to a significant improvement of the WOMAC score at 6 months, no significant change of cartilage defect	[81]
ADSC	Patients with III/IV symptomatic knee cartilage defect were intra-articular injected of AD-MSCs with fibrin glue and microfracture (MFX) treatment, a randomized study	Compared to the MFX-only group (control), ADSC injection in fibrin gel along with MFX improved KOOS pain and radiological symptom subscores, better tissue repair intensity	[82]
ADSC	Intra-articular injection of autologous ADSCs with low dose (2 × 106 cells), medium dose (10 × 106), and high dose (50 × 106) to patients with symptomatic primary knee OA and radiographic changes of grade 3 to 4 according to the Kellgren-Lawrence scale in the targeted knee OA randomized, double-blinded, and placebo-controlled study	No serious adverse events, well-tolerated in patients with improved WOMAC pain and function	[74]

Therapeutic applications of MSCs (BMSCs and ADSCs) are also being investigated in osteoporosis, characterized by loss of bone tissue increasing the risk of fracture, primarily in the aging population [83]. Preclinical studies in age-related osteoporotic mice showed that tail-vein injections of exogenous BMSCs increased bone quality, bone formation, and bone turnover rate in these mice [84]. Furthermore, in 2018, Uri et al. reported that in female ovariectomized rats, autologous ADSC-seeded scaffold implantation in the rat femur resulted in increased cortical thickness and bone volume density after 12 weeks. It also significantly increased femoral bone load to failure [85]. So far, two clinical trials have been conducted with patients with osteoporosis, one with intravenous BMSC injection (NCT02566655) and the other with ADSCs seeded in a composite graft to be transplanted into the patient’s fracture site (NCT01532076); however, the therapeutic outcomes are not clear or not reported.

From a clinical perspective, while the transplantation of adult MSCs has been successful, undesirable shortcomings and challenges should always be considered. Because of their ability to proliferate and resist apoptosis, the risk of tumorigenesis post–stem cell transplantation remains. Suzuki et al. reported that MSCs promoted tumor growth through neovascularization in murine models [86]. Other studies have shown that MSCs promoted tumor growth and metastasis in breast cancer [87], colon cancer [88], and pancreatic cancer [89]. De Boeck et al. reported that BMSCs drove and promoted tumorigenesis in colorectal cancer [90]. Furthermore, long-term in vitro MSC cultures pose a risk of chromosomal aberration, cytogenetic abnormalities, and genetic instability [91]. Depending on the route of administration of chosen cell lineage of MSCs, the biodistribution of the cells can be impacted and may get trapped in the lungs leading to undesirable effects and embolus formation [92]. For example, a 2015 study on pigs showed that compared to intravenous (IV) administration, intra-arterial (IA) of BMSC decreased lung entrapment and favored the accumulation of BM mononuclear cells in the liver and spleen avoiding undesirable pulmonary entrapment [93]. Finally, it is still challenging to have a sufficient number of desired adult stem cells, and usually, a very small number of cells in culture conditions are available, making it difficult to isolate and purify.

Engineered Stem Cell Classes

Class 3: Induced Pluripotent Stem Cells

The induced pluripotent stem cells (iPSCs) share similar properties to ESCs such as their ability for unlimited self-renewal and pluripotency. The main advantage of the iPSC over ESCs is that there is a considerably lower ethical barrier due to iPSCs being induced from mature somatic cells rather than a fertilized egg. By activating a class of genes in adult differentiated cells, these cells can be reverted to a pluripotent state [94]. Cells are reprogrammed into an embryonic stem cell–like state by forcing the expression of 4 transcriptional factors: octamer-binding transcription factor 4 (OCT4), sex-determining region Y-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and c-Myc (so-called OSKM factors) [3]. Due to their unlimited self-renewal and pluripotent qualities, iPSCs have been studied in a variety of regenerative engineering applications (Fig. 3). This review will focus on the use of iPSCs in musculoskeletal tissue regeneration.

Fig. 3.

Forcing expression of OSKM factors in adult somatic cells (such as fibroblasts and keratinocytes) induces dedifferentiation into a pluripotent, embryonic-like state. These induced pluripotent stem cells (iPSCs) can be differentiated into different lineages and used for autologous cell implantation

Due to its avascular environment, cartilage shows poor self-repair properties, and autologous tissue transplantation proves difficult due to the scarceness of donor tissue sites [95]. Tissue engineering approaches for the repair of articular cartilage face a major challenge of dedifferentiation of isolated chondrocytes after repeated passaging (therefore lowering regenerative outcomes) [95]. In one study, Zhu et al. successfully obtained iPSCs from human dermal fibroblasts with the OSKM factors and compared the regeneration of rat articular cartilage in a monosodium iodoacetate (MIA)-induced osteoarthritis (OA) model after injection of 500,000 differentiated or undifferentiated iPSCs. This study found that injecting iPSCs that were differentiated into chondrocytes and then injected had considerably better histological outcomes as well as reduced subchondral bone remodeling [96]. Uto et al. showed that GFP-labeled iPSCs in a collagen hydrogel could lead to cartilage regeneration [95]. They noted in a mouse osteochondral defect model that iPSCs without any filling could not renew the joint surface by 8 weeks, but by combining iPSCs into a collagen hydrogel, the joint surface was recovered in a dose-dependent manner. They further confirmed by immunohistochemistry that new chondrocytes were stained for GFP, indicating that new cartilage was formed from injected iPSCs [95]. In another study, Khan et al. demonstrated that depending on the origin of the donor, iPSCs isolated from either an OA donor (OA-iPSC) or a healthy donor (AC-iPSC) can retain distinct molecular memory and subsequently differentiate into chondrocytes using epigenetic and metabolism-specific transcriptional signaling pathways [97].

iPSCs have also been successfully used in tendon and muscle regeneration. Komura et al. observed that tenocytes differentiated from adult mouse iPSCs promoted Achilles tendon healing with minimal scar tissue formation compared to the carrier alone [98]. The authors further noted that the differentiated tenocyte expressed higher basic FGF (bFGF). The inhibition of scarring through higher paracrine secretion of bFGF may be a potential mechanism by which tenocytes derived from iPSCs drive regeneration [98]. This is especially important as it has been previously reported that adult tendon injuries do activate tenocytes but typically lead to fibro-cartilaginous tissue or ectopic bone formation at the injury site [99]. In a recent study, Tsutsumi et al. generated a tendon-like tissue called bio-tendon using human iPSC [100]. Here, iPSCs were differentiated into MSCs and subsequently transfected with Mohawk (MKX). These MKX-iPSC-MSCs were subjected to mechanical stretch in an incubation chamber system that resulted in bio-tendon. When transplanted in a mouse model of Achilles tendon rupture, an increase in cell infiltration improved histologic score with enhanced biomechanical properties [100]. In a separate study, Mizuno et al. successfully derived a cell population from mouse iPSCs that promoted muscular regeneration [101]. Grafted satellite stem cell marker (SM/C-2.6)-positive cells derived from mouse iPSCs showed significantly higher myofibers formation at 24 weeks compared to 4 weeks recovery time. In contrast, SM/C-2.6-negative cells derived from mouse iPSCs did not promote regeneration and instead resulted in the accumulation of inflammatory cells around the tissue [101]. Baci et al. identified that the source of iPSCs can play a role in their ability to differentiate into myotubes. They found that pericyte-derived iPSCs had a higher propensity to differentiate into myotubes compared to fibroblast-derived iPSCs when inducing differentiation with myotube-like extracellular vesicles in a muscular dystrophy murine model [102].

iPSCs are a great advancement in regenerative applications which also alleviate ethical concerns from ESCs. We have seen that the source of iPSCs impacts the extent of differentiation as well as the paracrine factors released from the differentiated cells. Recently, Guan et al. found a small molecule composition that can induce pluripotency from human somatic cells and that co-express OCT4, SOX2, and NANOG with an induction efficiency ranging from 0.21 ± 0.07 to 2.56 ± 0.63% [103]. While using small molecules to induce pluripotency in somatic cells remains highly desirable, matching the efficiency of forced transcription with small molecules remains challenging. As with ESCs, there is also a risk of teratoma formation with iPSCs. Cell integration into the tissue is critical for the therapeutic benefit, so an appropriate dose should be evaluated. Additionally, further characterization needs to be done to optimize cell sources and induction methods for specific tissues. Studies to compare differentiation ability and major paracrine factors released from these different cell sources of iPSCs should also be evaluated to progress iPSC-based therapy toward clinical translation.

Class 4: The Synthetic Artificial Stem Cell

One of the main mechanisms for how stem cells promote regenerative processes is their ability to secrete biological factors (secretome) that signal cell repair and regeneration. The composition of the secretome is highly dynamic depending on multiple factors such as the local microenvironment, physical cues, and extracellular matrix molecules. The secretome has been used as a cell-free alternative with several advantages for clinical use such as scalability, availability, and enhanced shelf life [60]. Previous synthetic approaches have been used to reproduce and deliver cellular components such as extracellular vesicles and the secretome through conditioned media [104, 105].

While the secretome poses many advantages over its cellular counterpart, this therapy still depends on isolating autologous tissue and cell expansion to obtain the conditioned media. Additionally, different secretome components may be redundant or antagonistic to specific tissue healing. For this reason, it is also beneficial to take a specific approach that can be used to tailor the paracrine effect and optimize specific tissue regeneration. Various materials such as biodegradable polymers have been developed to support endogenous regenerative ability and enhance tissue regeneration [106–111]. Regenerative engineering is defined as the integration of tissue engineering with advanced material science, physics, nanotechnology, stem cell science, developmental biology, and clinical translation [60, 112–117]. Therefore, the combinatorial use of nano-materials and biological factors can be one of the solutions for scalable and reproducible biological benefits [118, 119].

Circumventing the need for isolation and maintenance of cells, our group has engineered the synthetic artificial stem cell (SASC) using a recombinant secretome that has been formulated to mimic and tailor the paracrine effect on targeted tissue types (similar to how a biologically derived cell is a controlled delivery vehicle for the secretome) (Fig. 4). Growth factors identified to be key in the repair process were loaded into poly(lactic-co-glycolic acid) (PLGA 85:15) microsphere batches using a water-in-oil-in-water (W/O/W) double emulsion method after which microspheres in the 10–20 μm size range were isolated by sieving. Growth factor–loaded spheres can then be combined in different weight ratios to adjust the dosage of growth factors to tailor the regenerative potential to different microenvironments [120]. A controlled release of growth factors was used to effectively mimic the paracrine effect of stem cells. Furthermore, a direct injection rapidly clears from the delivery site; hence, a controlled release system prolongs the effect of the synthetic secretome for more efficacious therapeutic effects [60].

Fig. 4.

The synthetic artificial stem cell (SASC) system is designed to mimic the paracrine action of biologically derived stem cells to drive regeneration. Similar to how the ECM acts as a controlled delivery vehicle for the biological secretome (A), the SASC matrix controls the delivery of the loaded factors that are formulated for specific tissue regeneration (B). Previous studies have shown that SASC has similar cartilage regeneration potential compared to ADSCs and that SASC can be engineered to treat different disease models (C)

A prototype of the SASC system has been developed using osteoarthritis (OA) as a first model. Four growth factors, insulin-like growth factor-1 (IGF-1), transforming growth factor-β1 (TGF-β1), human growth hormone (HGH), and fibroblast growth factor-18 (FGF-18), were chosen as anabolic, chondroprotective factors to attenuate OA progression [120]. When challenged against collagenase-induced OA, the engineered SASC was shown to have similar regenerative potential to the biologically derived adipose-derived stem cells (ADSCs)-injected group. Furthermore, the cartilage that resulted in both these groups was mechanically superior to the disease control, indicating that the engineered SASC system may be a suitable alternative to cellular/cell-based therapy without the potential risk of biologically derived/semi-engineered class of stem cells such as tumorigenicity [120].

Due to the synthetic nature of the SASC, the paracrine effect can be fully engineered to fit different tissue systems and disease models. Furthermore, SASC cells provide clinical benefits over their biologically derived and semi-engineered counterparts. One such advantage is improved shelf life due to no cell viability challenges. Cell maintenance and expansion play a key role in other classes of stem cell–based therapies (ESCs, MSCs, and iPSCs), but the SASC cell is a microsphere-based system that can be manufactured on demand, negating the need for multiple patient visits that would consist of cell isolation and a follow-up visit for therapy (cell) injection. On the other hand, SASC cells may require higher doses to reach the same therapeutic potential compared to biologically derived counterparts. Since SASC cells do not proliferate or differentiate, the paracrine effect is dependent on a fixed particle quantity as opposed to cell-based systems in which therapeutic benefit additionally results from the stem cell’s ability to differentiate and proliferate to replace native tissue. Also, cell adhesion at tissue surfaces may increase retention of cell-based therapies, but in the SASC system, SASC would not attach to any surfaces leading to potentially lower retention. For this reason, it is critical to identify a proper SASC dose and/or engineer a system that would help retain SASC cells in a locally injected environment. For example, amnion-based hydrogels have been shown to enhance the retention of ADSCs while also providing an additive effect on the ADSC’s ability to attenuate collagenase-mediated OA cartilage degeneration [121, 122]. SASC may be loaded into a similar hydrogel system which can help retain SASC cells in the joint and lower the dose needed to maintain a therapeutic effect.

Conclusion and Future Directions

In conclusion, various innovations have made stem cells a highly sought-after disease-modifying therapy for regenerative engineering. Over time, we have collectively enhanced our understanding of these biologically based systems and used these insights to engineer specific stem cell functions. Each class of stem cells has its advantages and disadvantages (Table 2), but critically, with our knowledge of the mechanisms behind stem cell function, we have been able to engineer a completely synthetic system that effectively mimics biological effects (the SASC cell). The introduction of this system allows us to tailor paracrine responses to specific tissues with the ability to modify the composition of the secretome. As mentioned earlier, the uniqueness of SASC is it can be fully engineered and modified to fit different tissue systems and disease models, a major advantage over biologically derived stem cells. While SASC provides many clinical advantages, the integration of SASC cells into scaffolds and how these scaffolds interface together will play an integral role in applying SASC to complex tissue regeneration. Furthermore, now that we have completely synthesized an essential biological product, we must comprehend the pathology of a specific disease to identify suitable SASC compositions that effectively modify disease progression.

Table 2.

Advantages and disadvantages of stem cell classes

Stem cell class	Advantages	Disadvantages
Embryonic stem cells (ESCs)	Pluripotent potential Unlimited self-renewal	Ethical concerns surrounding cell isolation Low engraftment Tumorigenicity
Mesenchymal stem cells (MSCs)	Low immunogenicity No ethical concerns Ease of isolation Multipotent ability	Dynamic secretome composition dependent on various cues Limited cell viability Spontaneous changes in phenotype through multiple passages Tumorigenicity Limited cell number
Induced pluripotent stem cells (iPSCs)	Ability to isolate from abundant cell sources Unlimited self-renewal Pluripotent	Tumorigenicity Immunogenicity Low iPSC yields
Synthetic artificial stem cell (SASC)	Enhanced shelf life No viability or expansion limitations Composition not dependent on external factors Ability to engineer disease modification	No proliferative ability Limited retention in localized areas Higher doses or multiple injections required