Hematopoietic stem cells: what they are and where they are used

Hematopoietic stem cells are a rare population of cells that ensure the lifelong formation of blood cells and the restoration of hematopoiesis after injury. They are distinguished by two key abilities: self-maintenance of their own cell supply and multidirectional differentiation into erythrocytes, platelets, neutrophils, monocytes, T- and B-lymphocytes, and other mature cells. In healthy individuals, the majority of these stem cells remain dormant, awakening during blood loss, infection, or after myelotoxic therapy. This prevents depletion of the reserve while simultaneously maintaining stable hematopoiesis. [1]

Stem cell function depends on the microenvironment within the bone marrow—the so-called "niche." The niche is formed by endothelial and perivascular cells, osteoblasts, fibroblasts, macrophages, and neural elements, which signal stem cells to initiate dormancy or activation. Spatial atlases of human bone marrow have shown that different regions of the niche differentially support dormancy, division, and maturation, and inflammatory signals can shift the balance toward release from dormancy and subsequent exhaustion. [2]

With age, stem cells change their behavior: the proportion of cells with a "myeloid bias" increases, and their ability to rejuvenate and regenerate decreases. These changes are partly determined by intrinsic factors within the cell itself and partly by the "aging" of the niche. This explains why older people experience more cytopenias, have poorer recovery from chemotherapy, and have an increased risk of clonal mutations in hematopoiesis. [3]

The discovery and study of human stem cells has been accelerated by single-cell "cell atlas" technologies that map the transitions from stem cells to progenitors and mature lineages. New studies integrate transcriptomics, chromatin, and spatial data, helping to more accurately determine the signals that transition a cell from quiescence to division and into a specific lineage. [4]

Where and how do they appear in ontogenesis?

During human embryogenesis, hematopoiesis develops in waves. The first wave in the yolk sac produces early blood cells and tissue macrophages. Subsequently, in the aortogonadal-mesonephros region, precursors emerge from the hemogenic endothelium through the endothelium-to-blood junction. These precursors then migrate to the fetal liver, where they rapidly expand and mature. The final permanent site becomes the bone marrow, from where hematopoiesis is maintained throughout life. [5]

Recent studies have refined the markers of true hemogenic endothelium in humans. It has been shown that expression of the CD32 receptor in the endothelium of four- to five-week-old gestation cells identifies cells already irreversibly programmed for hematopoiesis. This helps model the early stages of hematopoiesis and identify conditions for producing fully functional stem cells in the laboratory. [6]

The fetal liver is a key "incubator" for stem cell expansion. Local liver cells and vascular elements create a factor-rich environment that enhances division and maintains immaturity. Spatiotemporal maps have shown that hepatoblasts and endothelium are the key "niche voices" that support a massive expansion of stem cell numbers in a short period of time. [7]

Interest in extraembryonic sources continues unabated: the placenta and its hemogenic endothelium, given certain signals, can be converted into cells with the characteristics of hematopoietic precursors. This is not yet a complete replacement for natural stem cells, but the field is rapidly progressing and is bringing us closer to the creation of safe, "personal" hematopoietic sources. [8]

How to recognize them: markers and functional tests

Identification of human stem cells relies on the rejection of "lineage" markers of mature cells and positive signs of immaturity. The highly enriched fraction in humans is most often described as "lineage-negative cells bearing CD34, lacking CD38, bearing CD90, and lacking CD45RA." A cell population with this phenotype yields the largest proportion of long-lived multidirectional regenerative potential in functional assays. [9]

It's important to note that the expression levels of individual proteins are context-dependent and can be confusing. For example, the surface protein CD90 is prominently detected on bone marrow stromal cells, and only accurate antibody panels and titers can avoid misinterpretations. Therefore, modern protocols utilize complex marker combinations and validation in transplantation models. [10]

Functional assessment includes "transplantation" tests in immunodeficient mice, colony formation in semi-solid media, and long-term cultures. Single-cell technologies make it possible to trace maturation trajectories from stem cells to progenitors and correlate marker phenotypes with the actual cell fate, increasing the accuracy of selection. [11]

Expanding stem cells outside the body remains challenging, but chemical and protein cocktails are emerging that allow for temporary self-renewal and reduce the loss of potential. Experiments in cord blood have shown that inhibiting certain pathways and precisely combining growth factors can increase the proportion of functionally competent cells after culture. [12]

Sources, acquisition, mobilization and cryopreservation

Stem cells are obtained from bone marrow, peripheral blood after "mobilization," and umbilical cord blood. Peripheral blood has become the dominant source in adults due to its faster neutrophil engraftment and ease of collection. However, chronic immune complications after peripheral blood transplantation are more common than after bone marrow or cord blood. The choice of source depends on the diagnosis, age, body weight, risk of relapse, and donor availability. [13]

Mobilization is the temporary "removal" of stem cells from the bone marrow into the blood. Granulocyte colony-stimulating factor is most commonly used, and in cases of risky or unsuccessful harvests, the CXCR4 receptor antagonist plerixafor is added. The choice of regimen depends on the prognosis of the "poor mobilizer," comorbidities, and the goal—autologous or allogeneic use. [14]

Cryopreservation using dimethyl sulfoxide (DMSO) allows transplants to be stored for years, but the cryoprotectant itself can cause infusion reactions, ranging from mild nausea to hypotension and bronchospasm. Reducing the DMSO concentration and washing the product out reduces these risks while maintaining viability, as confirmed by systematic reviews. [15]

For cord blood, the cell dose per kilogram of patient weight before thawing is critical. International recommendations specify a threshold of total nucleated cells of at least 2.5-3.0×10^7 per kilogram for one block in adults and allow the use of two blocks if volume is insufficient, with a target total level of at least 3.0-4.0×10^7. [16]

Table 1. Stem cell sources: strengths and weaknesses

Source	Pros	Cons	Typical application situations
Bone marrow	Lower risk of chronic immune reaction, better for some non-oncological indications	Slower neutrophil engraftment	Allogeneic transplants with a high risk of immune complications
Peripheral blood after mobilization	Fast engraftment, easy collection	Higher risk of chronic immune reaction	Most adult oncohematological patients
Umbilical cord blood	Rapid availability, less stringent antigen matching requirements, lower risk of severe chronic reaction	The cell dose is limited, engraftment is delayed	Lack of a suitable donor, low body weight, emergency cases

Based on comparative reviews and registers. [17]

When and what type of transplant is indicated

Autologous stem cell transplantation is used in multiple myeloma and some lymphomas to restore hematopoiesis after high-dose chemotherapy. In these situations, the stem cells themselves do not directly treat the tumor, but rather allow for safe, intensive antitumor treatment. [18]

Allogeneic donor transplantation is used for acute leukemia, myelodysplastic syndromes, aplastic anemia, and a number of hereditary blood diseases. Here, the therapeutic effect is derived from the replacement of hematopoiesis and the donor's immune "surveillance," which is capable of suppressing residual tumor cells. The choice of preparation regimen may be myeloablative or reduced-intensity, based on age and associated risks. [19]

For autoimmune diseases, immunoablative-prepared autologous stem cell transplantation is used in carefully selected patients as a way to "reboot" the immune system. Current guidelines emphasize strict selection and implementation in specialized centers, as the risks are no lower than for oncohematological indications. [20]

Gene therapy using autologous stem cells is a new reality for hemoglobinopathies. In December 2023, two different technologies were approved for sickle cell disease in adolescents and adults: editing to enhance fetal hemoglobin and adding a full beta-globin gene. In 2024, approval was also received in Europe, and in 2024-2025, indication expansion and refinement of monitoring programs will continue. [21]

Table 2. Types of transplantation and examples of indications

Type	The essence	Examples of indications	Key risks
Own stem cells	Return of purified autologous graft after intensive therapy	Multiple myeloma, lymphomas	Relapse of the underlying disease
Allogeneic stem cells	Complete replacement of hematopoiesis with donor cells	Acute leukemia, aplastic anemia, hereditary hemoglobinopathies	Graft-versus-host disease, infections
Gene therapy using one's own stem cells	Editing or adding a gene with the return of the modified cells	Sickle cell disease, beta thalassemia	Toxicity of preparation, unknown long-term effects

Key regulatory events and reviews. [22]

Compatibility and donor selection

A complete match for the five major antigens of the histocompatibility complex remains optimal. In the absence of a related "ideal" donor, unrelated donors with a match for the major alleles are successfully used, and controlled mismatches are also permitted using modern immune-complication prevention protocols. Donor selection takes into account age, gender, cytomegalovirus status, and other factors. [23]

In the era of post-transplant cyclophosphamide, the use of "partially compatible" and "nearly compatible" unrelated donors has expanded. This has increased the availability of allogeneic transplantation without significantly compromising survival with appropriate prophylaxis, as confirmed by modern registry analyses and clinical trials. [24]

For cord blood, a lower antigen match is permitted than for bone marrow or peripheral blood, provided the cell dose is sufficient. When selecting two blocks, the total cell dose and each individual unit are taken into account. These algorithms are formalized in guidelines and protocols. [25]

Fine-tuning of mismatches is gaining momentum: computer models allow the selection of "resolvable" mismatches associated with better relapse-free outcomes. This is especially important in transplants from an unrelated donor with a single mismatched antigen. [26]

Table 3. Donor selection priorities

Criterion	What is considered optimal?	Comment
Antigen matching	Complete match at five major loci	Minimizes immune complications
Donor age	A younger donor	Lower risk of chronic immune reaction
Source of cells	Selection based on diagnosis and risks	Peripheral blood - faster engraftment, but higher chronic immune reactions
Umbilical cord blood	Less overlap is acceptable with sufficient dosage	The key factor is cells per kilogram of mass

In total, according to modern guidelines and analyses. [27]

Prevention of immune complications and preparation regimens

The treatment regimen is selected individually. Myeloablative regimens are suitable for younger, more robust patients, while reduced-intensity regimens offer a chance for the elderly and weakened. In any case, the goal is the same: to create space in the bone marrow and suppress the immune system so that the donor cells can engraft. [28]

Standard prophylaxis for acute immune reactions includes combinations of a calcineurin inhibitor and methotrexate or mycophenolate. Since the introduction of post-transplant cyclophosphamide, the safety of transplants from partially matched donors has increased significantly, and outcomes are also improving with fully matched donors. [29]

A new player is abatacept, which blocks T-cell costimulation. In randomized and registry-confirming studies, the addition of abatacept to standard prophylaxis in unrelated and partially matched donors improved survival and reduced severe acute reactions. This is reflected in 2024 papers and center practices. [30]

In August 2024, axatilimab, an antibody to the colony-stimulating factor-1 receptor that targets abnormal macrophages and fibrosis, was approved for chronic immune response. This expanded the treatment armamentarium for patients who have failed two or more systemic therapies and is already scheduled for release in 2025. [31]

Table 4. Key approaches to the prevention and treatment of immune complications

Task	Approach	What is it based on?
Prevention of acute reactions	Calcineurin inhibitor plus methotrexate or mycophenolate	Decades of practice and meta-analyses
Expanding Compatibility	Post-transplant cyclophosphamide	Reducing the impact of mismatches without losing control over the disease
Strengthening prophylaxis in unrelated donors	Addition of abatacept to the standard	Clinical trials and registries for 2024
Treatment of chronic reaction after failure of two lines	Axatilimab	Regulatory approval in 2024

Summary data. [32]

Complications and how they are managed

The main early risks are infections associated with severe immunodeficiency, toxicity of the preparation, and delayed engraftment. The source of the transplant and the dose of cells influence the rate of recovery. Engraftment is slower in cord blood, but the risk of a severe chronic immune reaction is lower. [33]

Graft-versus-host disease (GVHD) can be acute or chronic. The acute form occurs in the first few months and affects the skin, liver, and gastrointestinal tract. The chronic form can involve multiple organs, requiring long-term immune therapy. Modern preventive and early treatment regimens improve tumor control without significantly increasing tumor recurrence. [34]

Infusion reactions to cryoprotectants are usually mild and transient, but require preparedness for management. Centers reduce the dimethyl sulfoxide load, use washout, and carefully planned premedication. This reduces the risk of nausea, hypotension, bronchospasm, and conduction disturbances. [35]

Long-term risks include recurrence of the underlying disease, clonal hematopoietic abnormalities, and cardiovascular events in the presence of clonal hematopoiesis in elderly patients. Accumulated data associate clonal hematopoiesis with an increased risk of cardiovascular complications, which is taken into account during monitoring and risk factor correction. [36]

Table 5. Frequent complications and preventive measures

Complication	When is it expected?	What reduces the risk
Infections	The first weeks before immune reconstitution	Barrier measures, antibacterial and antifungal prophylaxis
Acute immune reaction	The first months	Properly selected prevention and monitoring
Chronic immune response	Months and years	Source selection, modern targeted drugs
Infusion reactions to cryoprotectant	On the day of infusion	Reducing the concentration of dimethyl sulfoxide and washing

Brief summary of center-level approaches. [37]

Comparison of transplant sources: the nuances of choice

Collection from peripheral blood provides a high probability of neutrophil and platelet engraftment, which is critical under intensive regimens. However, cumulative series show a higher rate of chronic immune response than with bone marrow. Choosing bone marrow may be preferable to minimize chronic complications in unrelated donors. [38]

Cord blood allows for rapid treatment initiation in the absence of a compatible donor. Modern meta-analyses demonstrate comparable disease control with less chronic immune toxicity, but at the cost of delayed engraftment. Precisely calibrated cell dose thresholds are key to success. [39]

Registries clearly demonstrate that prophylaxis and preparation strategies alter the balance of risks. The addition of new prophylaxis options for unrelated donors is gradually eroding previous differences between sources, and the choice is increasingly dictated by specific clinical needs and logistics. [40]

Finally, the patient's age and weight, the rate of disease progression, and the availability of the center's resources often determine the final choice. For example, in smaller patients, cord blood is often optimal in terms of timing and safety. [41]

Table 6. Summary criteria for selecting a transplant source

Criterion	Peripheral blood	Bone marrow	Umbilical cord blood
Rate of engraftment	Fast	Average	Slowly
Chronic immune toxicity	Higher	Below	Below
Availability	Donor and mobilization required	Donor and puncture required	Bank, quick allocation
Effect of cell dose	Highly desirable, usually achievable	Hesitates	Critical, there are thresholds

Summary of reviews and registers. [42]

Innovation: From Induced Cells to Gene Editing

Directed differentiation culturing of human induced pluripotent stem cells allows for the production of cells capable of long-term hematopoietic reconstitution in animal models. Although translation to clinical use requires evidence of safety and stability, significant progress has been made and paves the way for "personalized" transplants. [43]

Editing one's own stem cells has already become a common practice for sickle cell disease and beta thalassemia. Approved products use different approaches, including increasing fetal hemoglobin by editing regulatory regions and adding a corrected copy of the gene. Patients undergo conditioning and the return of their own modified stem cells, after which crises and transfusion dependence can be eliminated or dramatically reduced. [44]

New biologic agents for chronic immune response, such as axatilimab, are changing the focus of late-stage disease management by targeting innate immune cells and fibrosis. Updates to drug formulations in 2025 reflect the rapid adoption of these approaches. [45]

In parallel, precise mapping of "niches" is being developed, which will help create artificial microenvironments for the safe expansion of stem cells outside the body. This is critical to overcoming the limitations of cord blood and reducing donor dependence. [46]

Brief summary

Hematopoietic stem cells are the foundation of hematopoiesis and a powerful therapeutic tool. In recent years, data on the role of the niche, age-related changes, and clonal hematopoiesis have become more robust. The clinic has expanded its donation options with post-transplant cyclophosphamide and abatacept, gene therapies have emerged, and a new target has been approved for chronic immune response. The choice of source and strategy should be based on the diagnosis, risk profile, and the center's infrastructure.