A typical preclinical trial of SC therapy for ED is schematically depicted in . It involves the isolation, cultivation, sorting, and modification of SCs, followed by labeling them with a cell-tracking agent. The labeled SCs were then injected into the corpus cavernosum (IC injection) of an ED animal model. Weeks or months later, the animals are tested for erectile function, usually by measurement of increases in intracavernous pressure (ICP) during electrostimulation of CN. The animals are then sacrificed for histological assessment of corpus cavernosum and tracking of injected SCs.
FIG. 3. A schematic representation of the experimental procedures of a typical preclinical stem cell therapy for ED. The donor rat and recipient rat can be the same (autologous) or different (allogeneic). The isolation and cultivation of SCs vary from one type (more ...)
Rat is the most commonly used animal in ED research and was used in all preclinical SC-for-ED studies thus far. Underscoring clinical needs, CN injury and DM are the most commonly tested disease models. CN injury, tested in 7 studies, was induced by either crush or resection of CN bilaterally. T2DM patients outnumber T1DM patients 9 to 1, yet most experimental studies chose T1DM over T2DM. This is due to the fact that T1DM can be easily induced by intraperitoneal injection of streptozotocin, whereas T2DM is more difficult to induce or requires the purchase of costly genetically modified animals. So, in SC-for ED field, T1DM model was used in 3 studies and T2DM in 1. Aging-related ED is largely manageable with PDE5 inhibitors, so SC studies in this category (a total of 3) were most likely motivated by not requiring any special treatment on the animal. Finally, hyperlipidemia-associated ED, which requires feeding animals with costly high-fat diet, was used in one study.
Types of SCs that have been used in experimental ED treatment (number of studies in brackets) are bone marrow (6), adipose (5), skeletal muscle (2), embryonic (1), endothelial progenitor (1), and umbilical cord blood (1, a clinical trial). Some studies have provided reason for choosing 1 particular SC type over the other; however, personal preferences and/or preexisting circumstances (eg, prior experience with a particular type of SC) probably played bigger roles than sound scientific rationales did. In any case, the pros and cons of various types of SCs can be found abundantly elsewhere and will not be discussed here.
Nearly all transplantations were done either autologously or allogeneically, the single exception being from mouse to rat. The transplanted cell number averages around 1 million per recipient rat. Most studies (total of 10) employed unmodified SCs, whereas others used SCs that were transfected or fractionated or in combination with other agents. Transfection or fractionation inevitably introduces risk factors (eg, virus) into the system and/or substantially reduces the number of treatment cells. Thus, knowing that the majority of studies employed unmodified SCs with satisfactory outcomes, the need to use modified or sorted SCs requires additional evidence.
A wide variety of methods have been used to monitor the distribution and survival of transplanted SCs. In SC-ED field, 2 studies by the same group of researchers transplanted human SCs to rats; therefore, cell tracking was done by the identification of human-specific nuclear protein. However, these studies did not assess erectile function and thus will not be discussed further. All other SC-ED studies used cells labeled with LacZ, 4’,6-diamidino-2-phenylindole (DAPI), green fluorescent protein (GFP), DiI (also known as PKH-26), bromodeoxyuridine (BrdU), or 5-ethynyl-2-deoxyuridine (EdU). Because the accuracy of data interpretation depends on the reliability of these labeling methods, potential problems are summarized below.
LacZ is a bacterial gene that encodes β-galactosidase (β-gal); however, many mammalian cells and tissues contain endogenous β-gal, making the detection of LacZ-transfected cells after their transplantation technically challenging [34
]. GFP is a protein from jellyfish. However, because of autofluorescence in mammalian tissues, GFP detection can seem like “seeing the wood through the trees” [35
]. DAPI binds to DNA noncovalently; therefore, it can leak from labeled cells after transplantation and be adsorbed by host cells, resulting in false-positive detection [36
]. DiI binds to cell membrane noncovalently and can leak from transplanted cells to host cells. In addition, because of DiI's cytotoxicity, transplanted cell preparations may contain debris of dead cells. Adsorption of this DiI-labeled debris by host cells can lead to false-positive identification [37
]. BrdU is incorporated into newly synthesized DNA and such labeled cells are detected with anti-BrdU antibody. However, the immnodetection of BrdU requires harsh treatment of tissue samples, resulting in distorted histological images [42
]. In addition, the denaturing treatment can cause loss of antigenicity of cellular proteins, making it impossible to detect cellular differentiation through immunohistochemical colocalization of cell type-specific protein. Even if the protein of interest survives the denaturing treatment, it is still difficult to identify the BrdU label with confidence, because its brown color cannot be easily distinguished from the purplish nuclear stain [43
]. EdU is a newer thymidine analog and is detected by a simple chemical reaction that requires no special tissue treatment [42
]. However, similar to BrdU, if a labeled cell is replicative after transplantation, its EdU label gets diluted with each round of cell division. So, long-term detection of transplanted cells is possible only if the cells are relatively quiescent. In our experience with EdU-labeled adipose-derived SCs (ADSCs), their detection in transplanted tissue is possible for at least 5 months after transplantation.
Stem cell transplantation
Before the introduction of PDE5 inhibitors, which are taken orally, IC injection of erectogenic agents was the most effective treatment for ED [4
]. Indeed, even nowadays patients who do not respond to or cannot take PDE5 inhibitors are still prescribed with IC injection of vasodilators. As this route of drug administration targets the organ of failure directly, it is commonly believed that IC injection is a locally applied intervention. However, we have observed that IC injected growth factors were able to restore erectile function through repair of damaged CN, whose cell bodies reside in the MPG [44
]. Further, in our first SC-for-ED study, we observed that intracavernously injected SCs could treat CN injury-related ED [13
], and all subsequent studies using different types of SCs confirmed the validity of this injection method for treating CN injury-related ED. Together, these data strongly suggest that IC injection is systemic in nature.
In all of our published SC-for-ED studies we reported difficulties in finding the transplanted SCs in penile tissues even though the animals clearly demonstrated functional and structural improvements [13
]. In studies published by others, intracavernously injected SCs were similarly difficult to find. In one of our studies we examined the presence of SCs in the penis at 2, 14, and 28 days after IC injection; the results clearly showed a time-dependent decline of the number of SCs [23
]. In our recent studies we conducted more definitive quantitative analyses and the results showed that the majority of intracavernously injected SCs exited the penis within 1 day [32
]. Further, in 1 of these 2 studies we showed that intracavernously injected SCs preferentially traveled to the bone marrow [32
]; in the other we found that intracavernously injected SCs also traveled to the MPG of CN injury rats, and this appears to be mediated by upregulated SDF-1 in the MPG [33
]. Together, these data suggest that (i) IC injection is essentially like intravenous (IV) injection—because of the fact that the cavernous sinusoids are essentially bundled venules (), (ii) the therapeutic efficacy of SCs for CN injury is due to SC trafficking to the MPG, (iii) systemically (IC or IV) applied SCs home-in to bone marrow, in support of the concept that mesenchymal SCs originate from bone marrow, and (iv) home-in of SCs to bone marrow may permit establishment of SC reservoirs for sustained regenerative and/or repair activities.
In animal experimentation, the most commonly used method for functional assessment of erection is measurement of intracavernous pressure (ICP) during electrostimulation of CN. This procedure requires laparotomy followed by sacrificing the animals; therefore, it is done near the end (most commonly at 1 month post-treatment) of a preclinical trial. As mentioned in the Rationale for Using SC Therapy section earlier, sexual stimulation triggers CN to release NO, which then causes CSMC relaxation and sinusoidal engorgement. In animal experiments, stimulation of CN with electric current mimics sexual stimulation and causes an increase of ICP that can approach systemic blood pressure, depending on the amperage of the applied electric current. Typically, at settings of 1.5
Hz, and pulse width 0.2
ms, the electrostimulation causes an increase of ICP (in cmH2
O) from a baseline of 20 to around 100 in normal rats. In ED rats, the rise is usually to around 30, and a successful SC treatment usually restores the value to about 70.
At the end of functional assessment, penile tissues are commonly prepared for examination by immunohistochemistry or immunofluorescence. The purposes of these examinations are to (i) locate transplanted SCs, (ii) correlate structural with functional changes, and (iii) identify possible SC differentiation. Localization of transplanted cells was discussed earlier under Cell labeling section. Assessment of structural changes invariably focuses on the 3 key components that regulate penile erection, ECs, CSMCs, and CN. ECs are commonly identified with antibodies against rat endothelial cell antigen, CD31, endothelial nitric oxide synthase, and/or von Willebrand factor (vWF). CSMCs are most commonly detected with anti-smooth muscle actin (anti-SMA) antibody. The most functionally relevant marker for CN is nNOS, as it identifies NO-releasing nerve fibers.
The concept of SC therapy was originally based on the premise that SCs have the ability to differentiate into various cell lineages. Thus, most SC therapy studies have strived to identify such events by checking whether the labeled SCs express cell type-specific proteins such as CD31 for ECs and SMA for CSMCs. So, it is obvious that the accurate identification of cell differentiation depends on the reliability of the SC trait/label, the differentiated cell marker, and the histological image. As discussed earlier in the Cell labeling section, with the exception of EdU, cell labels that have been employed in SC-ED studies cannot be detected with confidence. Moreover, histological images presented in most SC-ED studies are of low resolution and thus difficult to judge whether the so-called protein expression is indeed cellularly localized. In our experience, seemingly colocalized stains at low magnification often turned out not to be cellularly associated when viewed at higher magnifications (eg, 1,000×). Thus, it is crucial that claims of cell differentiation be backed by clearly discernable histological images.