Prenatal stem cell therapy for inherited diseases: Past, present, and future treatment strategies

Abstract Imagine the profits in quality of life that can be made by treating inherited diseases early in life, maybe even before birth! Immense cost savings can also be made by treating diseases promptly. Hence, prenatal stem cell therapy holds great promise for developing new and early‐stage treatment strategies for several diseases. Successful prenatal stem cell therapy would represent a major step forward in the management of patients with hematological, metabolic, or immunological disorders. However, treatment before birth has several limitations, including ethical issues. In this review, we summarize the past, the present, and the future of prenatal stem cell therapy, which includes an overview of different stem cell types, preclinical studies, and clinical attempts treating various diseases. We also discuss the current challenges and future strategies for prenatal stem cell therapy and also new approaches, which may lead to advancement in the management of patients with severe incurable diseases.


| POTENTIAL FOR PRENATAL STEM CELL THERAPIES
Many congenital diseases manifest early and thus prenatal diagnosis is often possible as early as 10-12 weeks of gestation. Our knowledge about the natural history of prenatally diagnosed disorders is often limited but at least in some cases it is known that damage continues to develop as gestation advances. Hence, there is a rationale for treatment to be initiated as early as possible, thereby ameliorating pathology or even preventing it from occurring. An improved situation for the fetus at birth might hypothetically benefit the transition from fetal to neonatal life and thus improve perinatal survival. Even though a single prenatal infusion may not be clinically sufficient for permanent improvement, a prenatal treatment approach is still justifiable since Other benefits of prenatal treatment include a better psychosocial situation for the mother and father from the birth of a child who has already been treated, and possibly an improved parent and family quality of life. Some of these benefits might also hold true for other types of inherited congenital disorders.
The use of high-resolution ultrasound has paved the way for early gestational diagnosis and cellular transplants and the procedure can be considered as safe, both for the fetus and the pregnant woman.
The prenatal infusion procedure is comparable to intrauterine blood transfusions, which are routinely performed in expert fetal therapy centers and where procedure safety (currently a procedure complication rate of 1.2% and a procedure loss rate of 0.6%) has improved significantly during the last 20 years due to improved transfusion techniques. 8

| STEM CELLS
Ever since Alexander Maksimov proposed the existence of stem cells in the beginning of the 1900s, 9 researchers have been searching for and identified stem cells from human tissue sources. There is a hierarchical capacity of self-renewal and differentiation ability of human stem cells ranging from the immature totipotent fertilized egg capable of forming all embryonic and extraembryonic tissues, the pluripotent embryonic cells that forms all cells in the developing fetus, the multipotent lineage restricted tissue residing stem cells, the oligopotent more restricted progenitor cells, to the mature unipotent precursor cells which only forms one cell type. 10 In addition to conventional stem cells, the work by Taka Table 1 together with their source and possible clinical potential.

| Totipotent stem cells
The first four cells in the embryo are totipotent and hence possess great potential, but are not eligible candidates for stem cell therapies and will not be further discussed here.

| Pluripotent stem cells
Embryonic stem cells (ESCs) are derived from the inner cell mass of the early embryo. Both the ESC and the iPSC in the immature pluripotent state form teratomas if transplanted and must be differentiated to a more mature and safe state before transplantation (Table 1 and

| Multipotent stem cells
Multipotent stem cells have good self-renewing potential but not to the same extent as pluripotent cells. Importantly, they do not form teratomas and do not necessarily need to be differentiated before transplantation.
Hematopoietic stem cells (HSC) derived from bone marrow (BM), mobilized into peripheral blood, umbilical cord blood (UCB), or fetal liver was the first stem cell type to be discovered, and is the most extensively investigated stem cell population, both experimentally and clinically. During the last 30 years, allogeneic HSC transplantation has become the major therapeutical approach for treatment of hematopoietic disorders such as leukemias, disorders of the immune system, and metabolic disorders and more than 40 000 transplantations using HSC are performed annually in Europe (www.ebmt.org). 12,13 After transplantation, HSC migrate to the BM, where they self-renew and reconstitute the defect hematopoietic system. White adipose tissue Adipose tissue is abundant in the human body and large amount of ADSC can easily be isolated with minimal donor site morbidity. The vast number of published preclinical studies of the ADSC reveals among other things the pro-angiogenic properties, and that the cells promote wound healing and tissue regeneration. 77 ADSC displays mesenchymal features but are more abundant and possess greater in vitro anti-inflammatory effects than bone marrow mesenchymal stem cell (BM-MSC). 77 These preclinical studies also provided evidence on the safety and efficacy of ADSC and several clinical trials regarding, for example, immune, orthopedic or soft tissue defects are currently ongoing. 77,78 Cardiac progenitor cells Heart tissue Fetal cardiac progenitor cells drive the growth of the developing heart through proliferation and possess regenerative properties. After birth both the proliferative and regenerative properties are diminished and the cells may exit the cell cycle. The existence of adult cardiac progenitor cells is controversial. Scientists discovering proliferative and thereby regenerative cells have most often detected DNA synthesis in polynucleated cardiomyocytes, which did not re-enter the cell cycle. 79 Postnatal c-KIT+ cardiac progenitor cells (CPC) have been reported to give rise to cardiomyocytes, smooth muscle cells and endothelial cells, and autologous c-KIT+ CPC has entered a phase I study while other studies suggest that 90%-100% of all of the cardiac c-KIT+ cells are actually mast cells. 80 For cell therapeutic purpose, cardiac progenitor cells seem unsuitable and other stem cells are being investigated, such as lineage-specified cardiopoietic MSC or stem cells differentiated from embryonic stem cell (ESC) or iPSC from heart fibroblasts. 81 Endothelial progenitor cells (EPC) Peripheral blood, spleen, vessel walls, and bone marrow EPC are matured from basal cells, and home to sites of vascular injury to restore vascular homeostasis and promotes neovascularization. After intracardiac injection of EPC in animal models of ischemia, blood perfusion was improved and intravenously administered autologous EPC increased cardiac function and reduced ventricular scarring after induced myocardial infarction, indicating promising therapeutic potential of the EPC. However, clinical studies with EPC as cellular therapy for ischemia could indeed present improved pathological features, although little or no clinical benefit could be observed. Therefore, potential clinical applications of EPC as cell therapy should await further safety, feasibility and efficacy studies before moving further toward the clinic. 82 Hepatic stem cells Liver tissue Hepatic stem cells have shown promising results as cell therapy for liver diseases when distributed via the portal vein. The cells homed and integrated into the lobes with cumulative decreased disease severity index (Mayo's Model for End-Stage Liver Disease) after stem cell distribution. The suggested source of stem cells is fetal tissues, as pediatric and adult livers are preferred as subjects for organ transplantation due to the constant lack of donor organs. 83 iPSC Somatic cells iPSC can in theory replace any pluri-or multipotent stem cell population for cell therapy and enables development of personalized treatment based on an autologous cell source. The challenge is to develop a robust differentiation method producing pure and uniform differentiated populations and to apply safety requirement to avoid teratoma formations. 84 Another concern is that the iPSC maintain the methylation state or epigenetic memory associated with their somatic cell-of-origin state which may influence their potential to differentiate into the cell type of interest. 85 (Continues) on the availability of family donors and worldwide registries with volunteer donors. UCB, on the other hand, is readily available from cord blood banks and complete HLA-matching is not needed. 13 However, one UCB unit may not be sufficient for an adult recipient, and infusion of donor lymphocytes is not possible due to the one single collection of a restricted volume of UCB.
Another promising multipotent cell is the mesenchymal stem or stromal cell (MSC). MSC can differentiate into mesodermal lineages, which is favorable in tissue reconstruction. MSC can be isolated from various tissues such as adipose, placenta (PL), umbilical cord (UC), UCB, amniotic fluid (AF), pre-and postnatal skin, and fetal liver, but the most common source is still adult BM. 14 MSC are minimally

Cell populations Sources Clinical potential and usability
Neural stem cells (NSC) Central nervous system NSC are rare populations in the brain and the access of human brain tissue is very limited. Previous isolations of viable NSC from human postmortem brain tissue showed reduced number of NSC with reduced proliferative capacity in the adult brains compared to prenatal brain tissue. 86 In contrast, NSC are more abundant in the developing early fetal brain and have been used in several clinical trials for treating neurodegenerative diseases. 87 To obtain a sufficient number of cells, multiple donors are required and hence with limited access to suitable donors and due to ethical considerations, scientists are considering using ESC or iPSC as an alternative source for replacement therapies for neurodegenerative diseases. 88  SCID is a unique disorder that provides a survival and proliferative advantage for donor T-cells, and the engraftment achieved in these patients has only been documented to reconstitute the T-cell lineage (split chimerism), 55 just as was observed in the early experimental work in mice performed by Blazar et al. 38 The results of the 46 clinical prenatal transplantation cases performed to date have clearly demonstrated that prenatal transplantation, using currently used methods, is not able to establish clinically relevant/ therapeutic levels of engraftment in recipients whose hematopoi-

| Present applications
Below, examples of preclinical investigations, case studies as well as clinical trials where prenatal cell therapy is an option for treatment of two selected diseases (Alpha-Thalassemia Major and Osteogenesis Imperfecta) are presented. utero. The rationale for using HSC from the mother is that the fetus will tolerate the mother's cells during pregnancy and the fetus will therefore not require any immune suppression. The trial will include 10 participants and is currently recruiting (ClinicalTrials.gov ID:

| Alpha-thalassemia major
NCT02986698 implying EVs as a promising therapy to prevent and treat perinatal brain injury. 75 The maintaining their biostability. Furthermore, since they are small and acellular, they might avoid entrapment in different organs, for example, the lungs and may potentially also be able to cross biological barriers. 76

| ETHICAL CONSIDERATIONS
One cannot underestimate neither the need for a well-controlled production of stem cells to be used as a medicinal product, nor the ethical considerations surrounding prenatal stem cell therapy and stem cell treatments. First, the source of the stem cells need to be well controlled so that the material is obtained with respect of ethical principles toward the donors. Furthermore, the cells need to be isolated, expanded, and quality controlled according to high-quality standards under Good Manufacturing Practice conditions, as would any Advanced Therapy Medicinal Product. Clinically, prenatal diagnosis must be accurate and reliable and must be communicated in such a way that the parents are able to make a well informed and well-founded decision of whether to agree to a prenatal stem cell therapy or not. Even though the outcome of a prenatal cell therapy may be very uncertain, the availability of a treatment before birth may affect the parents decision to continue or terminate the pregnancy. Lastly, one must keep in mind that apart from the fetus being treated, also the woman carrying the fetus might be affected by the treatment, further widening the need for a relevant ethical discussion.

| CONCLUSION AND LOOKOUT INTO THE FUTURE
Prenatal stem cell therapy, with all their in-built possibilities, can be a powerful tool to cure the incurable, already before birth. However, the limited clinical experience to date, often on heterogeneous case studies, means that it is not possible to be conclusive, and systematic clinical trials are vital to evaluate if a prenatal cell therapy approach is a realistic therapeutic alternative in specific diseases. In summary, establishment of prenatal stem cell therapy as a part of fetal therapy looks promising but bears several ethical and medical issues that must be addressed.